Microresonant sensors and methods of use thereof

ABSTRACT

A sensor assembly for sensors such as microfabricated resonant sensors is disclosed. The disclosed assembly provides improved performance of the sensors by providing a thermally insensitive environment and short pathways for signals to travel to processing components. Further, the assembly provide modular construction for the sensors and housing modules, thereby allowing replacement of the sensors at a lower cost. The assembly includes a sensor module including a sensor formed on a conductive substrate with a cavity formed on one surface. The substrate has conductive vias extending from the cavity to a second surface of the substrate. A housing assembly accommodates the sensor and includes a rigid housing, preferably made from a ceramic. An electronic component, such as an amplifier, is mounted on the rigid housing. The electronic component electrically engages the vias substantially at the second surface of the substrate. The electronic component receive signals from the sensor through the vias. The signals are then processed through an amplifier and a digital signal processor using a modified periodogram.

[0001] This application is a continuation in part of U.S. patentapplication Ser. No. 09/845,521, entitled MICROFABRICATED ULTRASOUNDARRAY FOR USE AS RESONANT SENSORS, filed Apr. 26, 2001; which claimspriority to U.S. Provisional Patent Application No. 60/233,961, filedSep. 20, 2000; and of International Application No. PCT/US00/29487,entitled MICROFABRICATED ULTRASOUND ARRAY FOR USE AS RESONANT SENSORS,filed Sep. 20, 2001, each of which is hereby incorporated by referencein its entirety, including all tables, figures and claims.

FIELD OF THE INVENTION

[0002] The present invention relates to sensors for monitoring a changein force as applied to a surface membrane or a change in the surfaceproperties of the sensor membrane. More particularly, the inventionrelates to a microfabricated mechanical resonant sensor and a sensorassembly.

BACKGROUND

[0003] The following description is provided to assist the understandingof the reader. None of the information provided or references cited isadmitted to be prior art to the present invention.

[0004] Technological advances in combinatorial chemistry, genomics, andproteomics have fostered an increased need for rapid high throughput(HTP) screening methods able to monitor and/or detect the reactionbetween one or more target species and binding partners or potentialbinding partners of such targets. Various systems have been, and arebeing, explored to detect analytes. Systems such as affinity chemicalsensing, arrayed sensors, and acoustic sensors are being investigatedfor their respective usefulness in detecting analytes in clinical andnon-clinical settings.

[0005] Affinity Chemical Sensing

[0006] Affinity chemical sensing systems attempt to detect interactionsbetween a target analyte and an appropriate binding partner. Suchsystems generally rely on the production or use of a detectable signal.Affinity chemical sensing systems employ binding partners which can bediscrete molecular species to which the target analyte specificallybinds, or a phase, such as an organic polymer, into which the targetpartitions. Covalently attached labels such as, fluorescent,electrochemical, radioactive, or mass based-probes are typicallyemployed in such systems. Methods for determining the presence analytesby using systems that detect the inherent optical, electrochemical, orphysical properties of a target species or changes in the properties ofthe layer containing the binding partner to which a target speciesbinds, have been employed to detect and/or monitor un-labeled analytes.

[0007] Charych, et al., U.S. Pat. No. 6,022,748, filed Aug. 29, 1997,describe an example of a sensor employing an optically active sensorcoating that changes color upon binding of the target. Further exampleof affinity sensing methods are described by W. Lukosz, “Principles andsensitivities of integrated optical and surface plasmon sensors fordirect affinity sensing and immunosensing”, Biosensors & Bioelectronics6, 1991, pp. 215-225. Utilization of surface plasmon resonance insensing applications is also described by Hanning in U.S. Pat. No.5,641,640, filed Dec. 29, 1994. A Chemically Selective Field EffectTransistor (CHEMFET) that determines target binding by monitoring asignal change on the sensor surface in response to target binding to thesaid surface, is described by Shimada in U.S. Pat. No. 4,218,298, filedNov. 3, 1978. Ribi et al., in U.S. Pat. Nos. 5,427,915 and 5,491,097,filed Aug. 9, 1993 and Feb. 28. 1994 respectively, describeaffinity-based microfabricated sensors in which a measurable change inconductivity of a bio-electric sensor layer is used to determine bindingof a target species.

[0008] Arrayed Sensors

[0009] Arrayed sensors have multiple individually addressable sites onthe device surface which are modified to contain binding partners for atarget molecule to be detected. An example of such a detection systemcan be found in U.S. Pat. No. 6,197,503, filed Nov. 26, 1997 by inVo-Dinh et al. The patent describes a device employing multiple opticalsensing elements and microelectronics on a single integrated chipcombined with one or more nucleic acid-based bioreceptors designed todetect optically labeled, sequence specific genetic constituents incomplex samples.

[0010] Other examples of arrayed sensors include: Pinkel et al., U.S.Pat. No. 6,146,593 filed Jul. 24, 1997, describe a method forfabricating biosensors using functionalized optical fibers to create ahigh density array of uniquely addressable biological binding partners;Fodor et al., U.S. Pat. No. 6,124,102 filed Apr. 21, 1998 describe anoptical sensor array having a planar surface derivatized with ligands ofan optically active target species immobilized at known locations suchthat each location comprises a “pixel” of an optical read out device.These and similar devices can be successful for arrayed detection andtherefore useful for parallel screening of multiple interactions wherethe analyte is either labeled or inherently optically, electrically, orspecifically chemically active.

[0011] Acoustic Sensors

[0012] Another field of technology having combine arrayed sensors isthat of sensors based on bulk or microfabricated resonant devices. Suchsensors have been demonstrated in systems used to determine3-dimensional acceleration, speed, and position, as transducers formonitoring environmental conditions such as pressure, fluid flow,temperature, and as gravimetrically sensitive elements in chemicalaffinity sensors.

[0013] Acoustic sensors for chemical sensing have been demonstrated inlow-density arrays in for example Ballato U.S. Pat. No. 4,596,697 filedSep. 4, 1984 which describes surface acoustic wave (SAW) devices. Arraysof cantilever sensors for gas phase sensing of multiple analytes aredescribed by Lang et al (Lang, H. P.; Baller, M. K.; Berger, R.; Gerber,Ch.; Gimzewski, J. K.; Battiston, F. M.; Fomano, P.; Ramseyer, J. P.;Meyer, E.; Guntherodt, H. J.; IBM Research Report, RZ 3068 (#93114),Oct. 19, 1998), and Britton et al (Britton, C. L.; Jones, R. L.; Oden,P. I.; Hu, Z.; Warmack, R. J.; Smith, S. F.; Bryan, W. L.; Rochelle, J.M.; Ultramicroscopy, 82, 2000, p. 17-21).

SUMMARY OF THE INVENTION

[0014] The invention described herein relates to sensors and sensorassemblies that can be used for various applications. The disclosedembodiments provide a sensor assembly that is tolerant to changes inenvironmental or system temperature changes. Further, the disclosedembodiments provide a sensor package that provides a modularconstruction, leading to a significant reduction in cost. Finally, theinvention provides for transmission and processing of signals from thesensor to electronic components, such as amplifiers, for more accurateand sensitive analysis. Current evanescent sensors known in the artrequire interactions to be very close to the surface. Acoustic sensorsdo not perform well in aqueous environments. Electronic sensors remainrelatively insensitive. The current patent discloses methods andcompositions that provide sensors, and preferably arrays of sensors,that perform well in water, have a good electrical interface, minimizeinterference, have a rapid and accurate signal processing scheme toallow sub Hz resolution of frequency shifts. The sensors of the presentinvention are particularly advantageous in biologic applications, whereaqueous environments are the norm.

[0015] The sensor in the package may be a microfabricated resonantsensors for monitoring a change in surface properties-of a sensormembrane that can be used individually or as an interconnected, yetelectrically isolated, grouping in microarrays. The change in surfaceproperties results from a binding event that changes the physicalcharacteristics of the membrane surface, such as surface mass, viscouscoupling, membrane stiffless, and the like. The sensors can also be usedto determine a change in force on the surface of a sensor membrane, suchas results from a binding event or application of pressure. A sensor canbe part of an array of sensors which can be fabricated to high density.The sensors and the sensor assembly of the present invention may havemany applications and may provide improved performance over existingdevices.

[0016] The term “sensor” as used herein relates to an apparatus ordevice that can respond to an external stimulus such as, a change inmass on a surface, pressure, force, or a particular motion, where theapparatus can transmit a resulting signal to be measured and/ordetected.

[0017] The term “binding event” refers to an interaction or associationbetween a minimum of two molecular structures, such as an analyte and abinding partner. The interaction may occur when the two molecularstructures are in direct or indirect physical contact. Examples ofbinding events of interest in the present context include, but are notlimited to, ligand/receptor, antigen/antibody, enzyme/substrate,DNA/DNA, DNA/RNA, RNA/RNA, nucleic acid mismatches, complementarynucleic acids, nucleic acid/proteins, and the like.

[0018] The term “analyte” or “target” refers to any molecule beingdetected by the sensor. The analyte (or target) is detected byimmobilizing one or more binding partners (or “probes”) or presumedbinding partners specific for the analyte or target to a sensormembrane. Thus, when it is desired to use the sensor to determine if agas or solution contains an analyte, the surface of the sensor membranethat is to contact the gas or solution is immobilized with a bindingpartner for that analyte. Analyte and its binding partner represent abinding pair of molecules, which interact with each other through any ofa variety of molecular forces including, for example, ionic, covalent,hydrophobic, van der waals, and hydrogen bonding, so that the pair havethe property of binding specifically to each other. Specific bindingmeans that the binding pair exhibit binding with each other underconditions where they do not bind to another molecule. Examples of typesof specific binding pairs are antigen-antibody, biotin-avidin,hormone-receptor, receptor-ligand, enzyme-substrate, lgG-protein A, andthe like.

[0019] Analytes or binding partners may be naturally occurring orsynthetically prepared. A “inatural analyte” is an analyte which occursin nature and specifically binds to a particular site(s) on a particularbinding partner such as a protein. Examples by way of illustration andnot limitation include a receptor and a ligand specific for the receptor(e.g., an agonist or antagonist), an enzyme and an inhibitor, substrateor cofactor; and an antibody and an antigen.

[0020] The term “antibody” refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0021] A typical immunoglobulin (antibody) structural unit is known tocomprise a tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively. An antibody can be specific for a particular antigen. Theantibody or its antigen can be either an analyte or a binding partner.

[0022] Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies, more preferably single chain Fv (scFv) antibodies in which avariable heavy and a variable light chain are joined together (directlyor through a peptide linker) to form a continuous polypeptide.

[0023] A single chain Fv (“scFv”) polypeptide is a covalently linkedVH::VL heterodimer which may be expressed from a nucleic acid includingVH- and VL-encoding sequences either joined directly or joined by apeptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci.USA, 85:5879-5883. A number of structures for converting the naturallyaggregated--but chemically separated light and heavy polypeptide chainsfrom an antibody V region into an scFv molecule which will fold into athree dimensional structure substantially similar to the structure of anantigen-binding site. See, e.g. U.S. Pat. Nos. 5,091,513 and 5,132,405and 4,956,778.

[0024] An “antigen-binding site” or “binding portion” refers to the partof an immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complimentarilydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987). An epitope is that portion of an antigen that interacts withan antibody.

[0025] “Sample” refers to essentially any source from which an analytecan be obtained. A sample may be acquired from essentially any organism,including animals and plants, as well as cell cultures, recombinantcells, cell components and can also be acquired from environmentalsources. Samples can be from a biological tissue, fluid or specimen andmay be obtained from a diseased or healthy organism. Samples mayinclude, but are not limited to, sputum, amniotic fluid, blood, bloodcells (e.g., white cells), urine, semen, peritoneal fluid, pleuralfluid, tissue or fine needle biopsy samples, and tissue homogenates.Samples may also include sections of tissues such as frozen sectionstaken for histological purposes. Typically, samples are taken from ahuman. However, samples can be obtained from other mammals also,including by way of example and not limitation, dogs, cats, sheep,cattle, and pigs. The sample may be pretreated as necessary by dilutionin an appropriate buffer solution or concentrated, if desired. Any of anumber of standard aqueous buffer solutions, employing one of a varietyof buffers, such as phosphate, Tris, or the like, preferably atphysiological pH can be used. A sample also my be artificially preparedsuch as a control sample that contains a known amount of an analyte.

[0026] Biological samples can be derived from patients using well knowntechniques such as venipuncture, lumbar puncture, fluid sample such assaliva or urine, or tissue biopsy and the like. Biological samples alsoinclude exhaled air samples as taken with a breathalyzer or from a coughor sneeze. A biological sample may be obtained from a cell or blood bankwhere tissue and/or blood are stored, or from an in vitro source, suchas a culture of cells. Techniques for establishing a culture of cellsfor use as a source for biological materials are well known to those ofskill in the art. Freshney, Culture of Animal Cells, a Manual of BasicTechnique, Third Edition, Wiley-Liss, N.Y. (1994) provides a generalintroduction to cell culture.

[0027] As used herein “microfabricated” refers to the procedures and/ormethods, such as bulk and surface micromachining, used to etch, deposit,pattern, dope, form and/or fabricate structures using substrates such assilicon and the like. Microfabrication procedures are known in the artand have been used to prepare Microsystems such as computer processorchips, acoustic sensors, micro-circuits and other devices requiringmicron and nanomolecular scale portions used in fields such asmicroengineering.

[0028] In one aspect, the present invention provides a micromechanicalsensor assembly for detecting, for example, a change in force at amembrane surface or a change in the surface properties of the sensormembrane. The sensor assembly of the invention comprises a sensor moduleincluding a sensor formed on a substrate. The substrate has conductivevias extending to a second surface of the substrate. The sensor assemblyfurther comprises a housing assembly adapted to accommodate the sensorThe housing assembly includes a rigid housing and is adapted toaccommodate an electronic component mounted on the rigid housing. Theelectronic component is adapted to electrically engage the viassubstantially at the second surface of the substrate. The electroniccomponent is further adapted to receive signals from the sensor throughthe vias.

[0029] As used herein, the term “module” refers to a portion of anassembly that may include a set of related components. A module may beinterchangeable with like modules.

[0030] The term “substrate” is used herein to refer to the startingmaterial from which the sensor of the invention is fabricated. Thesubstrate can comprise single crystal silicon, glass, gallium arsinide,silicon insulator, silicon-on-sapphire, and indium phosphate, and thelike. Also, combinations of these materials can be used. Preferably, thesubstrate has a high electrical resistance, such as a P or N-typesilicon wafer rated up to 15,000 Ω·cm. In a particular embodiment, thesubstrate comprises a silicon wafer, double side polished, P or N-typesubstrate having a resistance between 5 and 15,000 Ω·cm. Morepreferably, the substrate is a double side polished, silicon wafer, P orN-type having a resistance of roughly 10,000 Ω·cm. The substrate of thesensor can comprises one or more dopants, for example boron and/orphosphorus, to be patterned as one or more electrodes, and any vents,passages or holes within the cavity can extend through the substrate.

[0031] The term “via” is used herein to refer to a channel or a set ofchannels directed through a substrate. A via may be conductive ornon-conductive. Further, a via may be either filled with a material ormay be hollow. Vias may extend in any direction. Preferably, viasdescribed in this invention run substantially perpendicular to theplanar surfaces of the substrate.

[0032] As used herein, the term “housing” refers to an enclosure orsupport structure. Housing may be used to isolate components from theenvironment or to provide a stable environment for the components.Further, housing may simply provide a base for the components.

[0033] The term “rigid” is used herein to characterize the resistivetendency of a structure against vibrations, fluidity or other disruptivemovements. In one regard, rigid refers to tolerance to thermalvariations through low coefficients of thermal expansion.

[0034] The term “electrically engage” refers to an arrangement forallowing electrical communication. Components are generally electricallyengaged through physical interconnection.

[0035] In a preferred embodiment, the sensor comprises a membrane on thefirst surface enclosing the cavity and a second electrode spaced apartfrom the first electrode. The membrane includes a first electrode. Themembrane may be adapted to produce a membrane response when electricallyactivated.

[0036] The membrane of the sensor can be polygonal or elliptical. In apreferred embodiment, the membrane is rectangular having sides of 5 to10 microns in length. In another preferred embodiment, the membrane iscircular having a radius between 2 and 100 microns. The membrane coversa cavity in the substrate in a manner that prevents a fluid fromentering the cavity. Preferably, the membrane is up to 0.5 micronsthick. More preferably, the membrane is at least 0.05 microns and up to0.5 microns thick.

[0037] The cavity is preferably 0.1 to 2 microns deep and is sealed withthe membrane. Preferably, the cavity comprises one or more walls thatare 0.1 to 2 microns in height. More preferably, the cavity walls are0.3 to 1 micron in height, creating a cavity that is approximately 0.3to 1 micron deep. The height of the cavity is indicative of the distancebetween the electrodes of the sensor forming a capacitor.

[0038] The membrane or membrane layer of the sensor can be fabricatedfrom an electrically conductive material, such as doped single crystalsilicon, doped polysilicon, metal or any composite thereof, and canserve as a connection to ground. In alternative embodiments, themembrane can be fabricated out of non-conductive materials such assilicon nitride, silicon dioxide, phosphosilicate glass,borophosphosilicate glass. In this case, the membrane is not anelectrode but can have an electrode fabricated within, on, above orbelow the surface. As discussed herein, the membrane covers roughly theentire opening of the cavity in a substantially sealed manner. Themembrane of the sensor can also serve to conduct an electrical signal.In another embodiment the membrane layer can be fabricated to containone or more secondary structures that can conduct a current ofelectricity such as piezoelectric or piezoresistive materials. Inselecting a material to serve as a membrane for the invention sensor,certain mechanical characteristics such as Young's Modulus, which refersto the stifffiess of the membrane, the density, the intrinsic stress,and internal damping are considered. In a preferred embodiment of thepresent invention the membrane is prepared or fabricated in a mannerthat allows the membrane to vibrate and/or resonate. The membrane canalso be fabricated to either serve as an electrode for conductingelectricity, or as a connection to ground. The membrane can serve aspart of a capacitive or electrostatic pair. Within this embodiment, themembrane and the other electrode of the pair are separated by the spaceof the cavity and/or materials within the cavity, and act as a capacitorlike structure.

[0039] As used herein the term “membrane response” relates to thevibration or resonance of the membrane layer that is extended over, orplaced on, and roughly covers, in a sealed liquid impermeable, manner acavity of the invention sensor. Upon the introduction of a current orformation of an electrostatic potential, the membrane of the inventioncan move, vibrate or oscillate in a manner that can be measured, forexample, acoustically, electronically by electromechanical transductionsuch as by electrostatics/capacitance, piezoresistance orpiezoelectricity, or optically by interferometry, such as laser-Dopplervibrometery. The extent of vibration or oscillation of the membranedepends, for example, on the physical properties of the membrane and itsrelation to another electrode in the cavity or the effect of mass orforce on the membrane surface.

[0040] In a preferred embodiment, the rigid housing is formed of aceramic. In a more preferred embodiment, the rigid housing is formed ofa low-temperature co-fired ceramic (LTCC). In a further preferredembodiment, the housing is formed in layers of LTCC. In a still furtherpreferred embodiment, wire leads may be provided to extend from theelectronic component and through the layer of LTCC.

[0041] The electronic component may include stud contacts forelectrically engaging the vias. “Stud contacts” refers to protrusionsmade of conductive material that are adapted to electrically connect twocomponents. The stud contacts may be shaped to provide electricalcontact with a variety of components or may be customized to contact aspecific component. In a still further embodiment, the stud contacts aregold studs. These may interface with conductive polymer sheets such asZ-tape or Z-foam and allow reversible interconnection with electronicsbelow or may be permanently bonded to the electrical interface below.

[0042] The sensor module may further include a reservoir formed on thefirst surface of the sensor. The reservoir is adapted to retain a samplesolution therein. The sample solution may be a liquid or a gelcontaining a volume of sample to be tested, for example, for thepresence or absence of an analyte.

[0043] In one preferred embodiment, the sensor module includes anon-conductive frame for the sensor. The nonconductive frame at leastpartially forms the reservoir and is adapted to support the sensor onthe housing assembly. The nonconductive frame may be formed of aceramic. In a preferred embodiment, the frame is formed from an aluminacard, which may be provided with holes or cavities formed therein toaccommodate components or the reservoir.

[0044] The cavity in the substrate may be substantially evacuated andsealed. Evacuating the cavity may be performed in any number of knownways. The level of evacuation may be determined according to the desiredsensitivity of the sensor, for example.

[0045] The sensor assembly may also include sealable vent channelsextending from the cavity to the second surface for venting the cavity.The sealable vent channels may be used to evacuate the cavity, or may beused to occasionally vent the cavity to the atmosphere to normalize orcalibrate the operation of the sensor.

[0046] The vias in the substrate may include coaxial channels that maybe formed in a single hollowed channel and may be either insulated fromeach other or may be adjacent to each other. In a preferred embodiment,the coaxial channels include an outer conductive channel and an innerconductive channel The outer channel may be used for electricallyshielding the inner channel.

[0047] “Coaxial channels” refers to an arrangement wherein two or morechannels have a substantially similar longitudinal axis. In thisarrangement, the two channels may appear as, for example, concentriccircles in a cross-sectional view. Of course, cross-sectionalconfigurations other than circles may be used as well.

[0048] The electronic component may be one of many components, such asan amplifier. An amplifier may be used to boost a low-strength signalfrom the sensor so that it may be further processed and/or analyzed ormay be used to convert the high impedance of a small capacitive sensorto a lower impedance output to improve signal to noise ratios.

[0049] The sensor assembly may also be provided with a fluidics modulefor flowing a sample fluid to and from a sensor region of the sensor. Asensor region may be a surface area or a volume near, for example, amembrane of the sensor. The fluidics module may include a base supportedon the rigid housing. The base should generally be formed from amaterial having a low coefficient of thermal expansion, preferably aceramic and, more preferably, alumina.

[0050] The sensor module and the housing assembly may be modular andinterchangeable with other respective sensor modules and housingassemblies. In this regard, a sensor module may be made discardableafter a certain number of uses without the need to replace the entireassembly, which may include expensive electronics, for example.

[0051] In another aspect of the invention, a sensor element comprises asubstrate having at least one cavity formed on a first surface and amembrane on the first surface. The membrane encloses the cavity andincludes a first electrode. The sensor element further includes a secondelectrode spaced apart from the first electrode. At least one viaextends to a second surface of the substrate, the second surface beingopposite of the first surface. The via includes at least two coaxialchannels.

[0052] In a preferred embodiment, the coaxial channels include an outerconductive channel and an inner conductive channel. Alternatively, thecoaxial channels may include an outer conductive channel and an innerfluid channel. The inner fluid channel may be used to vent the cavity toatmosphere.

[0053] The coaxial channels may be separated by an insulating layer. Asused herein, insulating layer” refers to a layer of non-conductivematerial that may be deposited or grown on a surface. Suchnon-conductive materials may include a variety of materials such asoxides.

[0054] The cavity in the substrate may be substantially evacuated.“Substantially evacuated,” as used herein, refers to a low-pressureenvironment, preferably having a pressure of less than 1 T.

[0055] In another aspect, the invention provides a method of forming asubstrate for a sensor element. According to the method, a via from afirst surface of the substrate to a second surface of the substrate isformed. This may include electrochemically etching the substrate.Various methods of electrochemical etching are known to those skilled inthe art. The walls of the via are then coated with a first insulatinglayer. This may be accomplished by growing a conformal insulating layer.The conformal insulation is grown onto a surface to form a thin layer. Afirst conductive layer is formed over the first insulating layer. Thefirst conductive layer extends substantially from the first surface tothe second surface, and the conductive layer has a hollow, centralchannel therethrough. The first conductive layer may be formed byfilling a central region through the insulating layer with a conductivematerial, and etching the conductive material to form the hollow centralchannel therethrough.

[0056] In a preferred embodiment, the method further includes coatingthe walls of the hollow, central channel with a second insulating layer,thereby forming a hollow insulated channel through the first conductivelayer. In this regard, the hollow insulated channel is insulated fromthe first conductive layer. The insulated channel may be filled with aconductive material, thereby forming a second conductive channel throughthe via. Thus, two or more coaxial conductive channels may be formed inthe via.

[0057] In another aspect, the invention provides a method of processingsensor signals. According to the method, signals from a sensor aredigitized, and the digital signals are processed using a periodogram.The signals are responsive to a resonator that may drive a sensormembrane, for example. The periodogram is provided with a time-weightedcomponent adapted to weigh signal components according to asignal-to-noise ratio of said signal components.

[0058] A periodogram is a function for processing digital signals. Theperiodogram may be used to normalize the power spectrum for a signal orsignals in order to identify the signal in the presence of noise byestimating the power spectral density. The power spectral density is thedistribution of power over a range of the frequency spectrum. Thus, aperiodogram may output a frequency having a maximum power density,thereby identifying the signal frequency from the noise.

[0059] In a preferred embodiment, the time-weighted component is atime-decaying component. In this regard, because the signal is decayingwith time, the ratio of signal to noise is also decaying. Thus, atime-decaying component used for weighting the original samples willbias the output to the signal samplings with a higher (better)signal-to-noise ratio.

[0060] In a further preferred embodiment, the periodogram is defined by:${\frac{1}{N}{{\sum\limits_{n = 0}^{N - 1}\quad {{x(n)}\quad ^{{- \alpha}\quad n\quad T}^{{- }\quad \omega \quad n\quad T}}}}^{2}};$

[0061] where x(n) is an impulse response of said resonator, ω is angularfrequency, T is a selected sample period, and α is an empiricallydetermined constant. In this regard, the angular frequency may rangefrom 0 to 27π for the entire spectrum. By applying a typicaloptimization technique (e.g., golden-ratio optimization), one can applythe above formula at a small number of different frequencies andidentify the resonance frequency to approximately within 1 Hz.Empirically, it has been demonstrated that only the periodogram at fewerthan 30 frequencies need to be calculated to achieve the desiredfrequency resolution. With tighter manufacturing control, the number offrequencies where periodogram computation is required may be furtherreduced, and the computational load of the system can, therefore, alsobe reduced.

[0062] In another aspect, the present invention relates to methods foruse of resonant sensors to detect one or more binding events at or nearto the resonant membrane, and devices for performing such methods. Asdescribed in detail herein, the frequency of membrane resonance in suchsensors is sensitive to the mass attached to the membrane, as well as tochanges in mass in a geometric region above the sensor. While the exactshape of this region (e.g., spherical or parabolic) may vary, for thesake of simplicity, this shape may be approximated by a sphere having adiameter equal to that of the diameter of the membrane of the sensor.The area queried extends in the z axis dimension perpendicular to theplane of the membrane, and the sensor may be employed to sensedifferences in density within this region. As used herein, this conceptof density sensing is referred to as detection of an event “at or near”the resonant membrane surface.

[0063] While a device comprising a resonant sensor may comprise a singlesensor, in preferred embodiments the resonant sensor is formed into aresonant sensor array comprising a plurality of discretely addressableresonant sensors in a single physical device. Resonant sensor(s) can bearranged in an array from as few as a handful of sensor sites to as manyas 500,000 individual sensors/cm² High density arrays can comprisesbetween 256 to 150,000 individual sensors/cm² and more preferablybetween 5,000 to 100,000 sensors/cm². Each sensor in the array can beconfigured and arranged to detect the same binding event (e.g., eachsensor may comprise one or more binding partners for the same analyte ofinterest). However, in some embodiments, one or more individual sensorswithin an array can be configured and arranged to detect differentbinding events (e.g., one or more sensors in an array may comprise oneor more binding partners for a first analyte of interest, while one ormore different sensors in the array comprise one or more bindingpartners for a second analyte of interest). Preferably, an arraycomprises discretely addressable resonant sensors for the detection of2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 50000,100000, 200000, 500000, or more different analytes.

[0064] It is preferred that individual sensors sites are arranged in thearray in a manner that allows for electrical isolation of each sensor.In some embodiments, the individual sensor sites can be individuallyaddressed. In other embodiments, multiple sensor sites may be linked sothat they can be actuated and detected simultaneously.

[0065] By the term “addressable” when describing the electricalpotential of the sensor, membrane, electrode or an array, is meant thatthe described layer, sensor, substrate and/or membrane can accept anelectron, have an electric potential or voltage assignment. The electricpotential can be the assignment of having a ground voltage, such as forexample the membrane can be held at ground voltage when a sensoroperates using an AC, alternating current, power source, or theassignment can be a lower or higher electric potential within themembrane in reference to an opposing electrode if using a DC, directcurrent, electrode power source. The term “addressable” when used todescribe a sensor when placed in an array, combines the concept ofassigning an electric potential or voltage and relates to each sensorbeing capable of being given a specific locator and/or identifier,allowing a particular sensor in an array to be separately identifiablefrom surrounding sensors when used in methods such as high through putscreening.

[0066] Resonant sensors may also be formed into arrays having variousphysical dimensions and forms. For example, the sensors may be formedinto an array that is substantially circular, elliptical, square,rectangular, etc. Preferably, the sensors are formed into an arrayhaving a radius (if a circle) or axis (if an ellipse, square, orrectangle) that is between 200 μm and 200 mm, more preferably between500 μm and 100 mm, and most preferably between 1 mm and 50 mm.

[0067] In various embodiments, the resonant sensor(s) employed in thisaspect are those disclosed in U.S. patent application Ser. No.09/845,521, entitled MICROFABRICATED ULTRASOUND ARRAY FOR USE ASRESONANT SENSORS, filed Apr. 26, 2001; and International Publication No.WO 02/25630, entitled MICROFABRICATED ULTRASOUND ARRAY FOR USE ASRESONANT SENSORS, published on Mar. 28, 2002, each of which is herebyincorporated in its entirety, including all tables, figures, and claims.In particularly preferred embodiments, the resonant sensor(s) employedin this aspect are those described in detail herein.

[0068] In preferred embodiments, resonant sensors or resonant sensorarrays are used to detect the binding of a cell or vesicle at or nearthe resonant membrane surface. Cells, fixed or living, typically havediameters of 1-2 μm (bacteria), 7-10 μm (erythrocytes and white bloodcells) and 20-40 μm (tissue culture cells). Because they contain largeamounts of DNA (density 1.5g/mL), RNA (1.7 g/mL) and protein (1.3 g/mL),intact cells have a density greater than that of water: 1.09 g/cm³ forerythrocytes and 1.07 g/cm³ for white blood cells (with water having adensity of 1.0 g/cm³). Thus, cells passing near the sensor will bedetected as perturbations in density, causing the membrane to resonateat a lower frequency, without the need for any additional label.

[0069] Resonant sensors or resonant sensor arrays may be used forpurposes of “cell panning.” As used herein, the term “cell panning”refers to the binding of a cell to a surface due to specific binding ofthe cell to a binding partner for a cell surface epitope that isimmobilized on the surface. The array format described herein permitscell panning to be performed in parallel; that is, binding partners forone or more different cell surface molecules (e.g., epitopes, receptors,etc.) may be bound to different sensors in the array, and binding may bedetermined at each sensor in the array by detecting a change in resonantfrequency at a given sensor.

[0070] In various embodiments, array-based cellular panning may be usedto determine the phenotype of cells in a cell population. For example, aresonant sensor array may comprise one or more discretely addressableresonant sensors, each of which specifically binds a different cellsurface molecule. Preferred cell populations for use in such methods arelymphocytes, which may be screened for the presence or amount of cellsin each of a plurality of lymphocyte subsets (e.g., using antibodiesspecific for various CD or MHC markers immobilized to each resonantsensor); bacteria, which may be screened for the species and/or serotypeof organisms present (e.g., using species- or serotype-specificantibodies immobilized to each resonant sensor), and phage, bacteria, oryeast display libraries (e.g., using antigens of interest immobilized toeach resonant sensor). This list is not limiting, and the skilledartisan will understand that additional cell populations may be screenedby the methods described herein.

[0071] The term “phage display library” refers to a plurality of phage,each comprising a nucleic acid encoding an exogenous polypeptide fusedgenetically to a polypeptide directing expression of the exogenouspolypeptide on the phage surface. Typical commerically available phagedisplay libraries include structured peptide libraries, proteinlibraries, antibody libraries, linear peptide libraries, and enzymelibraries. Similar display technologies exist using organisms such asyeast and bacteria to display polypeptides of interest.

[0072] The skilled artisan will understand that such methods and devicesmay be advantageously used to screen for one or more compounds thataffect the phenotype of cells in a cell population. For example, samplesobtained from a human or non-human subject may be compared before orafter exposure of the subject to a treatment regimen for an effect onthe phenotype of cells present in the sample; alternatively, cells invitro may be contacted with one or more compounds being screened for theability to cause particular cells in a population to expand (or reduce)in number relative to other cells in the population.

[0073] Resonant sensors or resonant sensor arrays may also be used forpurposes of monitoring cell growth. The term “cell growth” in thiscontext refers to an increase in cell number at or near a resonantmembrane surface. In various embodiments, the resonant sensor orresonant sensor array may be used to detect cells growing free insolution (e.g., in a fermentor or in a roller bottle culture system) orcells growing on a surface (e.g., on the surface of a tissue culturesystem). In preferred embodiments, the sensor or sensor array may serveas a substrate surface upon which cells are grown. The term “substrate”in this context refers to a solid surface that acts as a support forcells. Cells may be grown directly upon this surface, or additionallayers (e.g., hydrogels, feeder layers, etc.) may be placed between thesurface and the cells.

[0074] In various embodiments, the resonant sensor may or may notcomprise a specific binding partner for the cells. In embodiments inwhich growth of cells in solution is monitored for example, growth inthe solution may be monitored by measuring the density of cells near theresonant sensor(s) (by detecting a change in resonant frequency at agiven sensor) without specific binding at the resonant sensor.Similarly, in those embodiments in which a resonant sensor or resonantsensor array is exposed to cells growing on the surface, a resonantsensor or resonant sensor array may provide a substrate on which thecells are grown. The extent of coverage of the array may be used todetermine the extent of growth; that is, as a cell colony expands ordecreases in size, changes in cell density at various portions of thearray may be monitored by detecting a change in resonant frequency at agiven sensor. The skilled artisan will understand that such methods anddevices may be advantageously used to screen for one or more compoundsthat affect the growth of cells.

[0075] Resonant sensors or resonant sensor arrays may also be used forpurposes of monitoring cell movements. The term “cell movements” as usedherein refers to the movement of one or more individual cells at or neara resonant membrane surface. In various embodiments, the ability ofvarious chemical or physical stimuli to induce or retard movement ofcells may be monitored. In such embodiments, a resonant sensor orresonant sensor array may provide a substrate on which cells aremaintained. The effect of one or more molecules (e.g., chemotacticmolecules; chemotactic inhibitors) on cell movement may be monitored bydetecting a change in resonant frequency at a given sensor.

[0076] Resonant sensors or resonant sensor arrays may also be used forpurposes of monitoring the binding of cells to ligands for cell surfacemolecules, such as receptors. In various embodiments, a library ofmolecules comprising a plurality of different molecular species to bescreened for the ability to bind to a particular cell surface moleculeare immobilized at one or more resonant sensors. Preferably, eachresonant sensor in an array comprises a different immobilized molecularspecies from the library. Cells expressing the cell surface molecule ofinterest are contacted with each resonant sensor, and the ability of oneor more cells to bind to a given sensor via receptor/ligand interactionis monitored by detecting a change in resonant frequency.

[0077] Similarly, one or more inhibitors of ligand binding may beidentified. In these embodiments, a library of molecules comprising aplurality of different molecular species to be screened for the abilityto compete with ligand for binding at a particular cell surface moleculeare immobilized at one or more resonant sensors. Preferably, eachresonant sensor in an array comprises a different immobilized molecularspecies from the library. Cells expressing the cell surface molecule ofinterest are contacted with each resonant sensor in the presence ofligand, and the ability of one or more cells to bind to a given sensorvia receptor/competitor interaction is monitored by detecting a changein resonant frequency.

[0078] In preferred embodiments, molecules that bind to a cell surfacemolecule may be ordered in terms of affinity for the cell surfacemolecule. In these embodiments, the number of cells contacted with aresonant sensor array may be reduced in steps, and the relative numberof cells bound to each immobilized molecular species determined as afunction of total cell number.

[0079] Preferred molecules for screening in such methods include smallmolecules, prodrugs, polypeptides, antibodies, antibody fragments,single-chain variable region fragments, polynucleotides,oligonucleotides, oligonucleotide analogs, oligosaccharides,polysaccharides, cyclic polypeptides, peptidomimetics, and aptamers.

[0080] As used herein, the term “small molecule” refers to compoundshaving molecular mass of less than 3000 Daltons, preferably less than2000 or 1500, still more preferably less than 1000, and most preferablyless than 600 Daltons. Preferably but not necessarily, a small moleculeis not an oligopeptide.

[0081] As used herein, the term “polypeptide” refers to a covalentassembly comprising at least two monomeric amino acid units linked toadjacent amino acid units by amide bonds. An “oligopeptide” is apolypeptide comprising a short amino acid sequence (i.e., 2 to 10 aminoacids). An oligopeptide is generally prepared by chemical synthesis orby fragmenting a larger polypeptide. Examples of polypeptide drugsinclude, but are not limited to, therapeutic antibodies, insulin,parathyroid hormone, polypeptide vaccines, and antibiotics such asvancomycin. Novel polypeptide drugs may be identified by, e.g., phagedisplay methods.

[0082] As used herein, the term “polynucleotide” refers to moleculecomprising a covalent assembly of nucleotides linked typically byphosphodiester bonds through the 3′ and 5′ hydroxyls of adjacent riboseunits. An “oligonucleotide” is a polynucleotide comprising a short basesequence (i.e., 2 to 10 nucleotides). Polynucleotides include both RNAand DNA, may assume three-dimensional shapes such as hammerheads,dumbbells, etc., and may be single or double stranded. Polynucleotidedrugs can include ribozymes, ribozymes, and polynucleotide vaccines.

[0083] As used herein, the term “oligonucleotide analog” refers to amolecule that mimics the structure and function of an oligonucleotide,but which is not a covalent assembly of nucleotides linked byphosphodiester bonds. Peptide nucleic acids, comprising purine andpyrimidine bases linked via a backbone linkage ofN-(2-aminoethyl)-glycine units, is an example of an oligonucleotideanalog.

[0084] The term “polysaccharide” as used herein refers to a carbohydratecomprising 2 or more covalently-linked saccharide units. An“oligosaccharide” is a polysaccharide comprising a short saccharidesequence (i.e., 2 to 10 saccharide units).

[0085] As used herein, the term “cyclic polypeptide” refers to amolecule comprising a covalent assembly of monomeric amino acid units,each of which is linked to at least two adjacent amino acid units byamide bonds to form a macrocycle.

[0086] As used herein, the term “peptidomimetic” refers to a moleculethat mimics the structure and function of an polypeptide, but which isnot a covalent assembly of amino acids linked by amide bonds. A peptoid,which is a polymer of N-substituted glycine units, is an example of apeptidomimetic.

[0087] The term “aptamer” as used herein refers to polynucleotides thatbind to non-polynucleotide target molecules (e.g., a polypeptide orsmall molecule).

[0088] While aspects and embodiments of the present invention aredescribed herein, it would be understood that such descriptions areexemplary of uses and aspects of the presently described sensors andarrays should not be limiting in content.

DESCRIPTION OF DRAWINGS

[0089]FIG. 1 is cross-sectional view of a sensor assembly according toan embodiment of the invention;

[0090]FIG. 2 is a cross-sectional view of an ultrasound sensor accordingto an embodiment of the invention;

[0091]FIGS. 3A to 3G illustrate a process according to an embodiment ofthe present invention by which coaxial channels are formed in vias;

[0092]FIG. 4A is a schematic illustrating the various capacitances whichmay exist in an electronic component receiving signals from the sensor;

[0093] FIGS. 4B-4D are schematic illustrations of embodiments of anelectronic component for processing of signals from a sensor such as thesensor illustrated in FIG. 1;

[0094]FIG. 5 is a graph illustrating standard deviation as a function ofQ for signals processed using a standard periodogram versus a modifiedperiodogram according to the present invention;

[0095]FIG. 6 is a graph illustrating standard deviation as a function ofthe sensor-specific constant alpha for use with a modified periodogram;and

[0096]FIG. 7 is a chart illustrating the capacitance as a function ofthe radius of a membrane.

DETAILED DESCRIPTION

[0097] The present invention is generally directed to a variety ofsensors, assemblies for such sensors and transmission and processing ofsignals from the sensors for analysis. The assemblies for the sensorsprovide improved performance of the sensors by providing a thermallyinsensitive environment and short pathways for signals to travel toprocessing components. Further, the assemblies provide modularconstruction for the sensors and housing modules, thereby allowingreplacement of the sensors at a lower cost.

[0098] Resonant sensors are disclosed in U.S. patent application Ser.No. 09/845,521, entitled MICROFABRICATED ULTRASOUND ARRAY FOR USE ASRESONANT SENSORS, filed Apr. 26, 2001; and International Publication No.WO 02/25630, entitled MICROFABRICATED ULTRASOUND ARRAY FOR USE ASRESONANT SENSORS, published on Mar. 28, 2002, each of which is herebyincorporated by reference in its entirety. The disclosed sensorcomprises at least two electrodes formed on a substrate having a cavity.Electrodes in the sensor cavity are preferably planar. The substrate maybe doped with an impurity which, depending on the type of substratechosen (p-type or n-type), can indicate either a substance such asboron, P-type, or phosphorus, N-type, to act as leads and/or electrodes.The electrodes of the sensor may also be formed of one or more metal ordiffused dopant electrode layers in the bottom of the cavity.

[0099] Leads are used to connect electrodes to a power source or ground.The leads can be prepared by fabricating conductive vias through thesubstrate cavity which lead away from the cavity in a substantiallyperpendicular manner. Such perpendicular leads can be prepared to extendthrough the substrate to the exterior of the sensor in order to beconnected to an electrical current source, or the leads can extend fromthe substrate cavity floor and be configured to exit the sensor at anangle, through one or more sides of the sensor itself.

[0100] Resonation or vibration of the membrane can be initiatedelectrostatically through use of electrodes in the sensor base, themembrane, the cavity wall, the cavity floor and/or membrane where theelectrodes are connected in a manner that allows the initiation orcreation of an electric current and/or potential. Resonation orvibration of the membrane of the sensor can be monitored usingelectrodes that can be located in and around the sensor as described andillustrated herein, and which can be part of a monitor apparatus, ormonitoring can occur, for example, either acoustically, electronicallyby electromechanical transduction such as by electrostatics/capacitance,piezoresistance or piezoelectricity, or optically by interferometry,such as laser-Doppler vibrometery.

[0101] Sensor or sensor arrays also can be used to determine known orunknown analytes in a sample using direct and indirect binding,competitive inhibition, sensitivity testing, specificity testing,affinity determination, and the like. For example, indirect binding maybe used when the amount of analyte that binds to the sensor membranesurface is too low for the sensor to detect. In this case, the sensorcan be contacted with a sample containing a binding partner specific forthe analyte bound to the sensor membrane. The sample-containing bindingpartner is preferably specific for site on the analyte that is separateand non overlapping from the site bound by the membrane immobilizedbinding partner such that the two binding partners can be boundsimultaneously to a single analyte molecule. Thus, indirect detection isachieved when the additional mass attributed to binding of thesample-containing binding partner to analyte on the membrane becomesdetectable. Competitive inhibition may be used with a sensor or sensorarray of the invention when an inhibitor analyte of lower mass inhibitsbinding of a larger mass analyte to the membrane.

[0102] Sensor Assembly

[0103] The present invention provides a sensor assembly for a resonantmicromechanical membrane sensor, for example, that is sensitive tochanges in the surface properties of the membrane surface such asdensity, inertia, viscous drag, or force. Measurement of a densitychange using the sensors of the present invention is particularly suitedfor the detection of molecular interactions in a gas or liquid phaseenvironment at the membrane surface of the sensor. A feature of thesensor is a drum-like cavity comprising a membrane at the top whichcontacts the environment to be sensed, or more walls that support themembrane, and a base with at least one electrode. The harmonic responseof the device is sensitive to the surface properties of the membrane.The membrane also protects the drive elements within the cavity fromdirect contact with the environment. The cavity also has other elementsand various sensor embodiments will now be described in detail.

[0104]FIG. 1 illustrates one embodiment of a sensor assembly accordingto the present invention. The sensor assembly 100 includes a housingassembly 102 and a sensor module 104 accommodated within the housingassembly 102. The sensor module 104 includes a sensor such as anultrasound sensor 106. The ultrasound sensor 106 is provided with aplurality of sensing regions, each region corresponding to a cavity suchas cavity 108. The cavities and the remaining structure of the sensors,such as sensor 106, are described below in further detail with respectto FIG. 2.

[0105] Referring again to FIG. 1, a conductive via, such as via 110, isprovided within the sensor 106. The via 106 leads from the cavity 108formed on the top surface of the sensor 106, to the bottom surface ofthe sensor 106. Each via 110 is adapted to conduct signals from thesensing regions above the cavities 108 to a signal processor. One methodfor constructing the vias 110 is described below with reference to FIGS.3A-3G.

[0106] The sensor module further includes a nonconductive frame 112. Thenonconductive frame may be formed of any of a number of nonconductivematerials. Preferably, the nonconductive frame 112 is formed from analumina card or other ceramic. In an alumina card, a region may be cutout to mount the sensor 106 therein. Ceramics such as alumina providethe advantage of extreme stability. For example, alumina has a very lowco-efficient of thermal expansion, thereby resisting changes due totemperature changes. In this regard, the sensor 106 remains stabledespite temperature changes in either the environment or in the samplesolution. Further, ceramics such as alumina resist torsion or otherdeformities in the configuration and, therefore, provide positionalstability for the sensors.

[0107] The nonconductive frame 112 may be attached to the sensor 106 ina variety of ways. In the disclosed embodiment, a nonconductiveunderfill 114 is provided around the perimeter of the sensor 106 tosecure the sensor 106 to the nonconductive frame 112. The underfill maybe made of any of a number of known materials that may function asinsulators, for example. Solder balls 116 may be provided within theunderfill 114 to provide a conductive path from the sensor 106 to leads(not shown) extending out of the sensor assembly 100. The leads mayprovide an electrical connection to a driving voltage for a membrane ofthe sensor.

[0108] Solder balls 116 may be shaped as barrels and may be embedded inthe underfill on all sides. A second underfill 118 may be provided abovethe nonconductive frame 112 to provide support and insulation foradditional modules.

[0109] The underfills 114 and 118 and the nonconductive frame 112together form a sample reservoir 120 above the sensor 106. The samplereservoir 120 may be adapted to hold therein a sample such as liquid ora gel containing materials to be tested. In this regard, the reservoir120 is electrically insulated from all electrical components, such asthe electronics for the sensor.

[0110] In one embodiment, the entire sensor module 104, including thesensor 106, the nonconductive frame 112 and the underfills 114 and 118,may be discardable. In this regard, the sensor module 104 and thehousing assembly 102 are made modular and interchangable with other likecomponents. For example, the same housing assembly 102 may be used witha variety of sensor modules such as sensor module 104. Thus, expensiveelectronics which may be included in the housing assembly 102 may beretained for reuse when a particular sensor module is to be discarded.

[0111] Housing assembly 102 includes a rigid housing 122 that may bemade of a rigid material, such as ceramic. In one embodiment, the rigidhousing 122 is made of a low-temperature co-fired ceramic (LTCC). Thisconstruction provides the housing with stability and resistance totemperature changes in the environment. Thus, a sensor module housed inthe housing assembly is stabley supported. As above, the ceramic or LTCCalso provides resistance to torsion or other deformities.

[0112] An electronic component 124 is mounted on the rigid housing 122of the housing assembly 102. The electronic component 124 may be anycomponent adapted to receive and/or process signals from the sensor 106.In one embodiment, the electronic component 124 is an amplifier forreceiving the signals from the sensor 106 and transmitting the amplifiedsignals to another processor. Alternatively, the amplifier may includecircuitry for digitizing the signals and performing digital signalprocessing.

[0113] Gold studs 126 are provided on the electronic component 124 toprovide electrical communication between the sensor 106 and theelectronic component 124. The gold studs may be shaped to interface anoff-the-shelf electronic component, such as an amplifier, to acustomized sensor such as a MEMS. In this regard, the cost of the sensorassembly 100 may be decreased through the use of cheaper off-the-shelfcomponents. In one embodiment, a silver filled epoxy may be used tocomplete the connection between the gold studs and the vias 110 of thesensor 106. Thus, the electronic component can be positioned adjacent tothe sensor 106, and signals from the sensor cavities 108 are directedthrough a path of minimal length through the vias 110. In thisconfiguration, unnecessary noise from an extended transmission path iseliminated. Signals are transmitted through the shortest path bytransmitting them directly from the sensor cavities 108 through the viasto an electronic component such as an amplifier for processing of thesignals.

[0114] The signals may be transmitted from the electronic component 124to additional electronics outside the rigid housing 122. In this regard,wire leads 128 are provided to extend from the electronic component 124.The wire leads 128 may extend through the rigid housing 122 to a regionoutside the sensor assembly 100. In one embodiment, the rigid housing122 may be constructed from layers of LTCC. The wire leads 128, then,may be directed between two layers through the rigid housing 122.

[0115] A fluidics module 130 may be provided to control the flow of asample fluid through the sample reservoir 120. The illustratedembodiment of a fluidics module 130 includes a fluidics base 132, whichis preferably formed of a rigid ceramic to provide thermal insensitivityand resistance to deformities. In one embodiment, the fluidic base ismade of either alumina or glass. The fluidics base 132 may be providedwith channels (not shown) for directing a fluid containing the sample toand from the sample reservoir 120. In this regard, a desired flow and adesired flow rate may be maintained within the reservoir 120. In theillustrated embodiment, an internal perimeter of the fluidics base 132combines with the underfills 114 and 118 and the nonconductive frame 112to form the reservoir 120.

[0116] The fluidics module 130 may also include a window assembly. Thewindow assembly includes a frame 134 which engages the fluidics base132. The window assembly also includes a window 136 that can bepositioned directly above the reservoir 120. The window 136 may be madeof several materials including glass or sapphire.

[0117] Thus, the sensor assembly 100 provides a rigid self-supportingstructure for the sensor operation. This self-supporting structure,preferably formed of a ceramic, allows a smaller package than may befeasible with other materials such as plastic. Further, the use of rigidmaterials such as ceramics inhibits vibrations or tortions of the sensorassembly, which may be a concern with plastic components. Further, themodular nature of the sensor assembly allows interchangeability of thevarious modules. As noted above, the sensor module 104 may be madediscardable, while the housing assembly 102 and the fluidics module 130may be reusable with other sensor modules. In this regard, a low-costsensor assembly is achieved.

[0118] Still further, the arrangement illustrated in FIG. 1 provides theimportant benefit of reducing unnecessary noise in the signals. Byproviding a direct path through the vias from the sensor region of thesensor 106 to an electrical component through a via 110, a significantreduction in noise that may result from an extended conductive path isachieved.

[0119] Sensor Structure

[0120] Referring now to FIG. 2, an embodiment of a sensor element thatmay be used with the sensor assembly described in FIG. 1 will bedescribed. The sensor element 200 is formed from a substrate 202. On onesurface of the substrate 202, one or more cavities 204 may be formed.Each cavity is provided with a membrane 206 that also either forms orincludes a first electrode for a capacitor. The first electrode may alsoserve as a lead to provide an input driving voltage to the membrane.

[0121] At the bottom of the cavity 204, a second electrode 208 isformed. Thus, the two electrodes 206, 208 in a spaced apartconfiguration form a capacitor. A sample solution may be provided abovethe membrane 206 and may include agents that may bind to the membrane206. When such binding occurs, certain properties of the membrane 206are altered. In this regard, characteristics such as the resonantfrequency of the membrane are also altered. Thus, binding events may bedetected through the detection of changes in resonant frequency of themembrane 206. In this regard, a driving frequency may be applied to themembrane 206 through an electronic source (not shown).

[0122] The substrate 202 is provided with a plurality of vias 210extending from the bottom of each cavity 204 to the bottom surface ofthe substrate 202. The structure of the vias and possible variationstherein are described below with reference to FIGS. 3A-3G.

[0123] In FIGS. 3A-3G, one embodiment of a process for forming the viasin the substrate is illustrated. A substrate 220 is etched to have a via222 formed completely therethrough (FIG. 3B). The via may be formed byany of a number of known etching methods such as laser etching usingmasks. Such methods are well known to those skilled in the art.

[0124] A conformal growth 224 is then deposited upon the substrate 220(FIG. 3C). The conformal growth 224 may be made of any insulatingmaterial such as a number of oxides. The conformal growth layer 224 isformed on all surfaces of the substrate 220 including the walls of thevia 222. A hollow channel through the via is maintained after theformation of the insulating layer 224 on the walls of the via.

[0125] Next, a conductive layer 226 is formed on the conformalinsulating layer 224 (FIG. 3D). The conductive layer 226 may be formedthrough several known methods including sputtering. In a preferredmethod, the conductive layer 226 is formed by first filling the via 222with a conductive material, which could initially be in the form of apaste. Once the via 222 has been completely filled with the conductivematerial, an etching process may be performed to create an openingthrough the conductive layer 226.

[0126] Next, a second layer of insulating material 228 may be formedthrough, for example, conformal growth (FIG. 3E). As above, the layer ofinsulating material 228 covers all surfaces including the walls of thevia. Again, a hollow channel through the center of the via is maintainedafter the formation of the insulating layer 228.

[0127] Next, as shown in FIG. 3F, a conductive material 230 is used tofill the hollow channel at the center of the via. The conductivematerial 230 fills the central region of the via and may also cover thetop and bottom surfaces of the substrate or conformal growth thereon.Finally, the layers of insulating material and conductive material areremoved from the top and bottom surfaces of the substrate (FIG. 3G).This may be performed in any number of ways including chemical cleaningor mechanical polishing.

[0128] Once the above-described process is completed, the substrate isleft with a via having a plurality of coaxial channels. In theembodiment illustrated in FIG. 3G, two coaxial conductive channels 232,234 are formed and are separated by insulation layers from each otherand from the substrate.

[0129] In another embodiment, the step described in FIG. 3F may beskipped. In this configuration, the completed substrate is provided withcoaxial channels including an outer conductive channel 234 and innerfluid channel, the hollowed central region of the via.

[0130] Thus, the formation of the coaxial vias can allow a sensormounted on one surface of the substrate to provide an electricalconnection to the opposite surface of the substrate. Thus, the sensor ontop of the substrate can send signals through the shortest path to anelectrical component on the bottom of the substrate.

[0131] Signal Detection and Processing

[0132] In a preferred embodiment, the system supports a 4×4 or 8×8 MEMSarray of disk diaphragm on silicon. The system design demonstratesrobustness to measure the resonant frequency shifts for single channel(1 Hz-100 Hz or so) and for scaling up the number of channels to a largenumber of channels. The MEMS designs provide the ability to: 1) enablelarge Q value in water, 2) allow electronic measurement of mechanicalmotion of the diaphragm by allowing it to be a significant percent ofthe gap between the electrodes (e.g., 40-160 nm for 1 um spacing fordriving voltage of 5-10V for 20 um radius), and 3) provide low straycapacitance.

[0133] The through holes (vias) are made using controlled porous silicon(e.g. coherent pores) by an anisotropic etching. The advantage of thisapproach relative to die-to-die wire bonding is in the fact that onecould achieve higher packaging density (3D stacking rather than 2D) and,thus, lower parasitics. Further, we can displace ASIC from the surfaceexposed to the chemo-biological solution environment to the bottomsurface of the substrate or COB.

[0134] In essence, the through holes may be arranged to be integral-viadesign to the MEMS chip. Ultimately the holes would be metallized withthe metal sleeve only, or may be filled with poly metal, and with lessthan 100 m-Ohms in-series DC resistance.

[0135] The theoretical model used to estimate transmission lineparameters may be based on quasi-TEM model, since the conductivity andthe dielectric between the through-hole contacts may be lossy. Theeffect of losses result in three major changes over the lossless case.The first change is that the forward and backward traveling waves sufferan attenuation as they move along the line. The second implication isthat the forward-traveling voltage and current waves may no longer be inphase since Zc is complex. The third change occurs in the velocity ofpropagation of the voltage and current waves.

[0136] The following was the standard set of lossydielectric-lossy-conductor formula used for this calculation:$\begin{matrix}{{{R\left( {\Omega/m} \right)} = {1/\left( {p\quad i*a*\delta*\sigma_{C}} \right)}};\left( {{\delta\bullet}\quad a} \right)} \\{{L\left( {H/m} \right)} = {\frac{\mu}{\pi}*\cosh^{- 1}\frac{d}{2*a}}} \\{{G\left( {{1/\Omega}*m} \right)} = \frac{\pi \quad \sigma}{\cosh^{- 1}\frac{d}{2a}}} \\{{C\left( {F/m} \right)} = \frac{\pi \quad ɛ}{\cosh^{- 1}\frac{d}{2a}}} \\{{where}:} \\{\delta = {\frac{1}{\sqrt{\pi \quad f\quad \mu_{C\quad}\sigma_{C}}} = {skin\_ depth}}}\end{matrix}$

[0137] where:

σ_(C), μ_(C), ε_(C)=ε₀

[0138] are conductor-specific resistance, magnetic permeability, anddielectric constant, respectively, and where:

σ, μ, ε

[0139] are characteristics of the dielectric that separates them.

[0140] The calculation above is applied on 10-, 15- and 20-μm diameterof the contact holes with oxide lined at the inner side (insulation) andfilled with a poly silicon (approximate Poly through hole fillerconductivity based on (0.0006 to 0.0050) Ohms-cm). All further specificexample calculations are based on value of 0.3 mOhms-cm.

[0141] The total thickness of the wafer used in calculations is 300 m.Bump calculation includes physical data parasitics gathered fromdifferent flip-chip and CSP/WSP packaging manufacturers (e.g., Kullicke& Soffa and Amkor Technologies).

[0142] Drum device radius design varies between 10-, 20- and 30-μm withthe approximate spacing of 1 um and membrane thickness of 1000-2000angstroms. Measurements have shown membrane displacement from 10-50 nmfor 20-40 Volts.

[0143] The initial spacing estimate does not include any external force(e.g., pressure differential or water pressure on the membrane) ornon-linearity of the restoring nor damping force. The distance betweenthe electrodes for the device in equilibrium could be found fromforce-balance equation or static capacitance measurements (providingthat we know, restoring force and electrostatic force). However, formodeling purposes, a simple parallel plate capacitance with one freeelectrode and one fixed electrode is used.

[0144]FIG. 7 shows capacitance as a function of radius of the membrane.As seen in FIG. 7, the equilibrium capacitance corresponding to the 10-,20- and 30-m radius are C10=2.78 fF, C20=11.12 fF, and C30=25.02 fF,respectively.

[0145] The bottom electrode may be highly n-doped silicon in 30 KOhm-cmhighly resistive n-type wafer pool. The preferred MEMS design electrodeis not insulated from the rest of the wafer electrically. This couldimply that instead of grounding the substrate, one could connect thesubstrate to the output where it would represent shield connected to theoutput of the buffer amplifier (in AC voltage divider topology). Thus,we would remove parasitics from the shield. This also would imply thatwe would have to ground bottom electrode instead of top and haveV_(drive) delivered to the top electrode.

[0146] If the bottom electrode is at the high potential, one would needto connect substrate (wafer) to the ground. However, the noisesusceptibility of the top electrode would be elevated since the groundedtop electrode would be affected by any electrical noise that is comingfrom the chemistry (polarization, electrical dipole effects and watercapacitor effects if any −tbd). Minimal electrical potential at thiselectrode would effect the measurements. Furthermore the top electrode(only 1000-2000 Angstrom's), if it is on a high potential and exposed tothe liquid, will require insulation relative to the liquid above. Thethickness of highly doped silicon membrane is way below the skin depththickness at the potential frequency of the operation and for theconductivity achieved by the doped silicon. Therefore, total shieldeffectiveness should be taken into consideration. In addition, whicheverend is grounded (the top or the bottom plate capacitor) should beincluded in parasitics.

[0147] Traditional low-capacitance sensors have used conventionalamplifiers with integrated buffers and op-amps. These amplifiers areeffective for sensors operating at relatively large sensing capacitance.However, many sensors, including sensors that may be used with thesensor assembly described above, operate at much lower sensingcapacitances of several ato-Farads (10⁻¹⁸), for example. At such lowsensing capacitances, parasitic capacitance well above the 1 pico-Faradlevel can attenuate the signal below the noise level. Further, manytraditional amplifiers require a larger footprint than may be desirablefor use in small sensors which may be placed in an array. The size ofthe amplifier may cause the sensors in the array to be more spaced apartthan either necessary or desirable.

[0148]FIG. 4A illustrates the various capacitances which may arise in anelectronic component such as a pre-amplifier or an amplifier. Inaddition to the primary capacitance being detected form the sensor,C_(S) 402, a reference capacitance C_(R) 404 is provided to provide abaseline for detecting the sensed capacitance. The reference capacitanceis generally selected to isolate the changes in the sensor capacitancein the presence of parasitic capacitances, as described below.

[0149] Parasitic capacitances may arise from several sources. Ingeneral, the addition of any electronic component may be accompaniedwith parasitic capacitances. For example, one parasitic capacitance,C_(P1) 406, may arise simply from the sensor itself. This may be causedby, for example, the flowing of the sample fluid above the membrane.Additional parasitic capacitance, C_(P2) 408, may arise from theimplementation of the electronic component itself. The interconnectionof the various components in a pre-amplifier or an amplifier (such asthose described below with reference to FIGS. 4B-4D) may cause aparasitic capacitance to arise. Finally, a particular type of parasiticcapacitance, called a Miller Capacitance, or C_(F) 410, may be intrinsicto certain components such as MOSFET gates.

[0150]FIG. 4B is a schematic illustration of one embodiment of anamplifier for use with the sensors described above. In this embodiment,the drive signal for the membrane, which may be substantially greaterthan the response signal from the membrane itself, is separated using adual-gate (MOSFET), or a lateral DMOS, and a phase-shifted drive signal.A dual gate FET, or MOSFET, is desirable for use in this arrangement dueto its lower input capacitance relative to conventional amplifiers.Since the signals from the sensors are typically low-capacitancesignals, this characteristic of MOSFETs improves the ability to processa measurable signal.

[0151] In this configuration, a drive signal is generated by an ACvoltage driver 412. The drive signal drives the sensor capacitance 411to generate a sensor capacitance signal that is indicative of theresonant frequency of the membrane. The signal that is output from thesensor (line 416) is a the response signal superimposed on the drivesignal. In this regard, the drive signal is substantially greater inamplitude than the response signal. Thus, even a high signal-to-noiseratio may be insufficient to isolate the response signal.

[0152] The drive signal from the voltage driver 412 is also transmittedto a synchronous demodulator 417, which shifts the signal by 180degrees. The synchronous demodulator 417 may be implemented in a varietyof ways including through the use of a conventional phase-locked loopwhich is adapted to generate a signal that is 180 degrees out of phase.Thus, the output signal from the demodulator 417 is exactly 180 degreesout of phase with the drive signal and is transmitted through line 419.

[0153] A dual gate MOSFET 418 or a lateral DMOS is provided to receivethe signals from the sensor capacitance 411 and the demodulator 417. Thedual gate directly mixes the two signals and outputs the response toanother component or sub-component for analysis (line 420). Thus, thelarge drive voltage component is exactly removed from the signal,providing only the membrane response signal. The response issubstantially similar to the membrane response.

[0154] In an alternative embodiment, one can adjust the signal level ofthe demodulator signal to maximize the membrane response signal. Becauseof variations between different sensors and variations during operation,the desired demodulator signal level can be automatically determinedeither by a feedback circuitry or by a microprocessor. This circuitryeffectively serves as an automatic gain controller (AGC), and canfacilitate maximization of signal quality.

[0155] In another embodiment, shown schematically in FIG. 4B, thepre-amplifier 421 compensates for parasitic capacitances and Millercapacitance through bootstrapping. In this arrangement, an AC voltagedriver 422 is provided to drive the membrane of the sensor 424. Areference capacitance 426 is again provided to facilitate isolation ofthe response signal. The output response from the sensor is directed toa dual gate MOSFET 427 or a lateral DMOS. Thus, at the point labeled “A”in FIG. 4C, the signal is the response signal from the sensorsuperimposed on the drive signal from the voltage driver 422.

[0156] At the point labeled “C” in FIG. 4C, the signal at point “A” isalso directed to a phase compensation stage 428. The phase compensationstage 428 includes a DC voltage drive 430 for driving a transistor 432.The signal from the transistor 432 is directed to the dual-gate MOSFET427 or a lateral DMOS through the point labeled “B”. The phasecompensation stage 428 then functions to maintain an identical signal atthe point “B” as at point “A” though gate-to-source bootstrapping. Thus,the output signal is phase-matched to the sensor response signal.

[0157] An impedance matching stage 436 may also be provided to improvethe amplitude matching of the output signal to the sensor responsesignal. The impedance matching stage illustrated in FIG. 4C includes aresistor 438 and a capacitor 440 through which a portion of the sensorresponse signal superimposed on the drive signal is directed.

[0158] Further, a Miller capacitance compensation stage 434 may beprovided to facilitate compensation of the Miller capacitance in thesignal. The Miller capacitance stage 434 is more clearly illustrated inFIG. 4D. As illustrated in the schematic illustration, the Millercapacitance stage is again gate-to-source bootstrapped through the phasecompensation stage 428. In this regard, this configuration may beconsidered to be double bootstrapped and is capable of compensating allparasitic capacitances, including the Miller capacitance resulting fromthe use of the dual-gate MOSFET 427.

[0159] Accordingly, the embodiments of the electronic componentdescribed with reference to FIGS. 4B-4D can provide a signal thatcontains no parasitics with the drive signal removed. Thus, the outputof the component includes a sensor response signal with little or noparasitic components. Combined with the short path length from thesensor to the component, a signal with low noise level can be achieved.

[0160] Alternatively, the membrane can be actuated with a narrow pulsewith a pulse duration significantly smaller than the period of themembrane vibration. Because the membrane typically vibrates for manyperiods, this actuation mode naturally separates sensor response to thedrive signal.

[0161] The response received from the sensor with pulse excitation isgenerally a transient response which may be sampled at a high frequency.In his regard, the determination of the resonant frequency may bedifficult from a low-Q sensor that may be used in the above-describedarrangement. In these cases, the power of the response signal may varygreatly over the sampled time. As a result of the varying power, thesignal-to-noise ratio also varies greatly.

[0162] Existing analysis algorithms for use with such sensors requirecomputation of a correlation matrix to isolate the resonant frequency.Calculation of the matrix, however, can be very intensive andtime-consuming, particularly where large data samples are gathered.Further, many existing algorithms are unable to isolate a second peak inthe power-frequency profile of the received signal. In other words,these algorithms may be unable to identify the proper resonant frequencyif another similar-powered frequency is present.

[0163] Periodograms are a search technique based on discrete Fouriertransform of input or measured values. The classic periodogram isdefined as a discrete sum:${\frac{1}{N}{{\sum\limits_{n = 0}^{N - 1}\quad {{x(n)}\quad ^{{- }\quad \omega \quad n\quad T}}}}^{2}};$

[0164] where x(n) is an impulse response of the resonator, ω is angularfrequency, and T is a selected sample period.

[0165] The above periodogram formula allows one to estimate the power ofa signal at a specific frequency. By using a typical optimizationtechnique (e.g., golden-ratio optimization), one can identify themaximum frequency of the capacitive sensor precisely with theapplication of the above formula at only a small number of differentfrequencies. This feature compares favorably with some of the “global”techniques that calculate the entire power spectrum.

[0166] Periodograms are generally not recommended for applicationsrequiring high spectral resolution due to lack of accuracy. However,since the present embodiment seeks to detect only the differences in theresonant frequency, not the resonant frequency itself, the desiredcharacteristic is precision, not accuracy. In fact, periodograms provideextremely good precision for resonant frequency calculations.

[0167] However, with a greatly varying signal-to-noise ratio, the noiselevel may sometimes have a bigger impact on the periodogram calculationthan the actual signal. In other words, if only a portion of themeasurement period contains the response signal and the remaining periodcontains either noise or extraneous signals, the measured responsesignal will be outweighed by the other components in the finalperiodogram sum. For example, the impulse response for a low-Q resonatorquickly sinks below the noise floor. Thus, the signal at the earliertime is a better indicator of the resonant frequency than the latertime.

[0168] A solution to this problem is achieved by including atime-dependant component to the classical periodogram. For example, theperiodogram may be modified as follows:$\frac{1}{N}{{{\sum\limits_{n = 0}^{N - 1}\quad {{x(n)}\quad ^{{- \alpha}\quad n\quad T}^{{- }\quad \omega \quad n\quad T}}}}^{2}.}$

[0169] In this manner, a time-decay component is added to theperiodogram. The time-decay component provides a weighting to thesamplings as signal-to-noise ratio deteriorates. In the above equation,α is a constant that may be empirically determined.

[0170]FIGS. 5 and 6 illustrate the effectiveness of the modifiedperiodogram noted above. FIG. 5 shows comparisons between analyses usinga classical periodogram (Alpha=0) and a modified periodogram with an αof 1.25. An effective measure of spectral analysis is the standarddeviation achieved. Ideally, a very low standard deviation is desired.As shown in FIG. 5, the modified periodogram outperforms the classicalperiodogram for all values of Q. In fact, the advantages of the modifiedperiodogram over the classical periodogram become particularlysignificant at low Q values.

[0171] Although the selected value of α should be empiricallydetermined, FIG. 6 demonstrates that the value need not be preciselycalculated. The standard deviation resulting from a modified periodogramanalysis does not substantially change over a large range of values ofα. For example, between cc values of 0.75 and 1.25, the standarddeviation achieved is substantially constant.

[0172] Applications for Use of Resonant Sensor Devices

[0173] As described in U.S. patent application Ser. No. 09/845,521,entitled MICROFABRICATED ULTRASOUND ARRAY FOR USE AS RESONANT SENSORS,filed Apr. 26, 2001; and International Publication No. WO 02/25630,entitled MICROFABRICATED ULTRASOUND ARRAY FOR USE AS RESONANT SENSORS,published on Mar. 28, 2002, resonant sensors and resonant sensor arraysmay be used for monitoring a change in surface properties of a sensormembrane resulting from a binding event that changes the physicalcharacteristics of the membrane surface, such as surface mass, viscouscoupling, membrane stiffness, and the like.

[0174] The frequency of membrane resonance in such sensors is alsosensitive to changes in mass in a geometric region above the sensor. Asnoted above, the shape of this geometric region may be approximated by asphere having a diameter equal to that of the diameter of the membraneof the sensor. Thus, the area queried extends in the z axis dimensionperpendicular to the plane of the membrane, and the sensor may beemployed to sense differences in density within this region. As aresult, the quantity of material passing near the sensor may be detectedby a change in resonant frequency of the sensor membrane.

[0175] For non-affinity based sensing, the density of particles comingwithin the sensed volume can be monitored without a need for surfaceimmobilization. The relative number or density of cells coming withinthis sensed volume could be used to monitor cell growth, either insolution (e.g., in a fermentor) or in cells requiring surfaceattachement (e.g., using the sensor or sensor array as a substrate toprovide the required attachment support. In these embodiments, aresonant sensor or sensor array may be used to identify molecules thathave an effect (positive or negative) on cell growth. For example,bacteria, tumor cells, normal cells, etc., may be grown on the surfaceof a sensor array, in which local regions of the array comprise growthenhancing or retarding compounds embedded in a hydrogel layer overlyingthe resonant sensor membrane(s). Selective growth would confer a densityshift which would be detected by the sensor. Similarly, the chemotacticeffects of factors immobilized to the surface of the sensor may bemonitored by detecting changes or gradients in concentrations of cellsdrawn selectively to a given sensor. Such methods could be used toidentify or assay chemokines or mitogens for example, or in woundhealing models. Such sensors may also serve as mass sensors for anelectrophoretic or chromatographic separation methods to providecontinuous real time sensing over a broad area, such as might be used ineither capillary electrophoresis or 1- or 2-dimensional electrophoresis.

[0176] The skilled artisan will recognize that such methods are notlimited to biological applications, and that the “deep query” feature ofresonant sensors and sensor arrays may be used generally as a densitysensor for liquid compositions. For example, particulate material insolutions could be monitored in manufacturing processes, in waste watertreatment, in plant exhaust gas emissions, in automotive emissions, orin slurrys. Likewise, changes in density in fluids caused bynonparticulate changes (e.g., changes in salinity or total dissolvedsolids) can also be monitored generally.

[0177] The term “dense particle” as used herein refers to a particlethat has a density greater than that of the surrounding fluid medium,while the term “buoyant particle” refers to a particle that has adensity less than or equal to that of the surrounding fluid medium. Theterm “liquid environment” as used herein refers to freely flowing liquidenvironments and any medium comprising water molecules within a solid orsemisolid matrix. Examples of non-freely flowing liquid environmentsinclude gels (e.g., polyacrylamide, agarose, starch, hydrogel, etc.),cell interiors, chromatographic media, etc. This list is not meant to belimiting.

[0178] For affinity based sensing, a binding partner for a molecule ofinterest must be immobilized either at the resonant membrane surface, orsufficiently near to the surface so that a binding event will result ina change of density within the sensed region. Numerous methods have beendescribed for immobilizing a molecule on a surface. For example, aphysical interaction that provides a direct contact of the molecule ofinterest with the surface, such as adsorption, can be used.Additionally, a chemical interaction that results in ionic or covalentcross-linking of the molecule to the surface can also be used. Forexample, U.S. Pat. No. 4,284,553, which is hereby incorporated byreference, including all tables, figures and claims, discloses methodfor the covalent immobilization of protein molecules to oxide surfacesvia thioester-containing coupling chains.

[0179] Alternatively, the molecule of interest may be indirectlyimmobilized on the solid surface. See, e.g., U.S. Pat. Nos. 6,171,610;6,156,572; 6,048,548; 6,039,977; 5,902,603; 4,452,892, each of which ishereby incorporated by reference, including all tables, figures andclaims; which describe methods of indirect immobilization, for example,by “enmeshing” or physically embedding a hydrogel comprising themolecule into a support surface, such as mesh cloth, or porous orroughened surfaces.

[0180] Molecules of interest in the methods described herein may includesmall molecules, polypeptides, proteins, cyclic polypeptides,peptidomimetics, aptamers, antibodies, scFvs, polysaccharides,receptors, polynucleotides, and/or polynucleotide analogs; and mayinclude therapeutic drugs, pathogens, biological agents, environmentaltoxins, etc.

[0181] As used herein, the term “small molecule” refers to compoundshaving molecular mass of less than 3000 Daltons, preferably less than2000 or 1500, still more preferably less than 1000, and most preferablyless than 600 Daltons. Preferably but not necessarily, a small moleculeis not an oligopeptide.

[0182] As used herein, the term “polypeptide” refers to a covalentassembly comprising at least two monomeric amino acid units linked toadjacent amino acid units by amide bonds. An “oligopeptide” is apolypeptide comprising a short amino acid sequence (i.e., 2 to 10 aminoacids). An oligopeptide is generally prepared by chemical synthesis orby fragmenting a larger polypeptide. Examples of polypeptide drugsinclude, but are not limited to, therapeutic antibodies, insulin,parathyroid hormone, polypeptide vaccines, and antibiotics such asvancomycin. Novel polypeptide drugs may be identified by, e.g., phagedisplay methods.

[0183] As used herein, the term “antibody” refers to an immunoglobulinmolecule obtained by in vitro or in vivo generation of an immunogenicresponse, and includes both polyclonal, monospecific and monoclonalantibodies, and antigen binding fragments thereof (e.g., Fab fragments).An “immunogenic response” is one that results in the production ofantibodies directed to one or more proteins after the appropriate cellshave been contacted with such proteins, or polypeptide derivativesthereof, in a manner such that one or more portions of the proteinfunction as epitopes.

[0184] As used herein, the term “single-chain variable region fragment”or “scFv” refers to a variable, antigen-binding determinative region ofa single antibody light chain and antibody heavy chain linked togetherby a covalent linkage having a length sufficient to allow the light andheavy chain portions to form an antigen binding site. Such a linker maybe as short as a covalent bond; preferred linkers are from 2 to 50 aminoacids, and more preferably from 5 to 25 amino acids.

[0185] As used herein, the term “polynucleotide” refers to moleculecomprising a covalent assembly of nucleotides linked typically byphosphodiester bonds through the 3′ and 5′ hydroxyls of adjacent riboseunits. An “oligonucleotide” is a polynucleotide comprising a short basesequence (i.e., 2 to 10 nucleotides). Polynucleotides include both RNAand DNA, may assume three-dimensional shapes such as hammerheads,dumbbells, etc., and may be single or double stranded. Polynucleotidedrugs can include ribozymes, ribozymes, and polynucleotide vaccines.

[0186] As used herein, the term “polynucleotide analog” refers to amolecule that mimics the structure and function of an polynucleotide,but which is not a covalent assembly of nucleotides linked byphosphodiester bonds. Peptide nucleic acids, comprising purine andpyrimidine bases linked via a backbone linkage ofN-(2-aminoethyl)-glycine units, is an example of an oligonucleotideanalog.

[0187] The term “polysaccharide” as used herein refers to a carbohydratecomprising 2 or more covalently-linked saccharide units. An“oligosaccharide” is a polysaccharide comprising a short saccharidesequence (i.e., 2 to 10 saccharide units).

[0188] As used herein, the term “cyclic polypeptide” refers to amolecule comprising a covalent assembly of monomeric amino acid units,each of which is linked to at least two adjacent amino acid units byamide bonds to form a macrocycle.

[0189] As used herein, the term “peptidomimetic” refers to a moleculethat mimics the structure and function of a polypeptide, but which isnot a covalent assembly of amino acids linked by amide bonds. A peptoid,which is a polymer of N-substituted glycine units, is an example of apeptidomimetic.

[0190] The term “aptamer” as used herein refers to polynucleotides thatbind to non-polynucleotide target molecules (e.g., a polypeptide orsmall molecule).

[0191] The term “therapeutic” or “drug” as used herein refers tomolecules or compositions used in the treatment or prevention ofdisease.

[0192] The term “pathogen” as used herein refers to agents that causedisease, including bacteria and viruses.

[0193] The term “biological agent” as used herein refers to one or moremolecules obtained from an organism.

[0194] The term “environmental toxin” as used herein refers to one ormore molecules that is a poisonous to one or more functions of a cell,and that are present in the terrestrial environment.

[0195] U.S. patent application Ser. No. 10/306,506 (Atty Docket No.035300-0502, filed Nov. 26, 2002), which is hereby incorporated byreference in its entirety, describes methods and compositions for theimmobilization of molecules at a surface in a biologically relevantaqueous or semi-aqueous environment, such as a water-swellable hydrogel.These methods allow molecules within the hydrogel to freely interactwith other molecules, and can mimic a biological environment. Onceimmobilized to a surface via a hydrogel, the molecule(s) can be used asan immobilized binding partner. Such an immobilized molecule can, forexample, be contacted with a test sample to determine if the samplecontains a binding partner for the immobilized molecule. The methods andcompositions can also be used in a microscale environment, such as atthe surface of a resonant sensor membrane.

[0196] Affinity based resonant sensors and sensor arrays may be employedin methods analogous to numerous analyte binding assays well known tothose of skill in the art. These include competitive and noncompetitive(e.g., sandwich) immunoassay formats, nucleic acid hybridization assays,etc. Advantageously, detection of a binding result is determineddirectly; that is, a change in mass or density at or near the resonantmembrane resulting solely from analyte binding to an immobilizedaffinity partner is sufficient to cause a detectable change in resonantfrequency. However, amplification methods may be employed to enhance thechange in resonant frequency. For example, an analyte bound to animmobilized affinity partner may be further contacted with a secondaffinity partner to form an affinity partner-analyte-second affinitypartner “sandwich.” The second affinity partner may be conjugated to amass enhancement moiety, such as a latex or metallic (e.g., gold)micro-or nano-particle, or an enzyme that catalyzes the deposition of aprecipitate. Enzymes that are useful for this amplification purposeinclude glucose oxidase, galactosidase peroxidase, alkaline phosphatase,and the like. Further, enzymes that elongate the detected species, suchas polymerases for DNA may be used. In particular, nucleic acidamplification methods such as strand displacement amplification, rollingcircle amplification, isothermic methods such as “nucleic acid basedsequence amplification (“NASBA”), or polymerase chain reaction (“PCR”)may be used. These methods may be used as anchored or non-anchoredprocedures. The addition of this additional mass within the sensedvolume can provide an enhanced resonance signal in the device.

[0197] In addition to the detection of the presence and/or amount ofanalytes such as proteins, small molecules, nucleic acids, etc., the“deep query” property of resonant sensors and sensor arrays allows theincorporation of 3-dimensional hydrogels onto the surface of thesensor(s) and/or allows the measurement of changes in the local densityof microparticles such as vesicles or cells. The ability to detectbinding interactions on the surface of intact cells has many advantages.Membrane embedded proteins (e.g. 7 transmembrane-family receptors or ionchannels), are difficult to extract from membrane while still preservingnative function. This occurs for several reasons. The proteins usuallyrequire a lipid component to retain native conformation and it isdifficult to devise a solubilization protocol which preserves this,requiring optimization for each receptor. Further, part of thespecificity of these receptors is derived from protein-proteininteractions which only occur in the native membrane. For the binding ofa transmembrane receptor to its ligand may be increased 100 fold or moreby association with its normal heterotrimeric G-protein partner. It hasalso been suggested that numerous receptors may function asheterodimers.

[0198] The array format enables the determination of cellular attachmentto and dissociation from immobilized molecules to be performed inparallel. Moreover, the ability to directly sense binding reactionsallows real-time monitoring of cellular attachment and dissociation forone or many cells on an individual sensor surface. This capability canbe applied to characterizing cell populations, e.g., for diagnosticpurposes. For example, A/B/O antigens on erythrocytes could be rapidlyidentified in a blood sample to determine blood type. Likewise, thepresence, absence, or amount of various CD antigen-bearing cells in asample could be rapidly determined. In such examples, antibodies foreach antigen of interest may be immobilized at discrete sensor locationsin a sensor array. Cells expressing a corresponding antigen bind to theantibodies result in a change in resonant frequency at a particularsensor location. The following tables provides an exemplary list of CDand other antigens for use in such methods: TABLE 1 Exemplary CDantigens Desig- nation Cells Expressing Function CD1 Dendritic reticularcells, Langerhans cell histiocytosis, few lymphoblastic lymphomas CD2 Tcells, NK cells LFA-3 (CD58) ligand CD3 T cells T cell antigen receptorstruc- ture, signal transduction CD4 Helper T cells MHC class IIcoreceptor, HIV receptor CD5 T cells, B cell subset, CLL, T cellactivation, CD72 ligand mantle cell lymphoma CD7 T cells, NK cells,early T and NK cell activation myeloid cells, some AMLs CD8 Cytotoxicand suppressor T MHC class I coreceptor cells, NK cells CD10 Early Bcells Neutral endopeptidase CD11b Monocytes, granulocytes Cell adhesionmolecule, part of CD11/CD18 integrin CD11c Granulocytes, monocytes, Celladhesion molecule, part hairy cell leukemia of CD11/CD18 integrin CD13Myeloid cells Aminopeptidase N CD14 Monocytes CD15 Granulocytes,Reed-Sternberg Lewisx antigen, cell adhesion cells, endothelial cellsand phagocytosis CD16 NK cells, granulocytes Fe gamma RIII (IgGreceptor) CD19 B cells B cell activation CD20 B cells Ca++ channel, Bcell activa- tion CD22 B cells Cell adhesion molecule CD23 Activated Bcells, CLL Fe epsilon RII (IgE receptor) CD25 Activated T cells,activated B Interleukin 2 receptor alpha cells, hairy cell leukemia,chain ATL/L CD28 T cells Delivery of second signal during T cellactivation, B7 (CD80/CD86) ligand CD29 Activated T cells Cell adhesionmolecule CD30 Activated T cells, Reed- Growth factor receptor Sternbergcells, anaplastic (similar to TNF receptor) large cell lymphoma, germcell tumors CD33 Myeloid cells Sialic acid adhesion molecule CD34Progenitor cells CD38 Lymphoid progenitor cells, Leukocyte activationplasma cells CD40 B cells Growth factor receptor, B cell activation CD41Megakaryocytes gpIIb, cell adhesion molecule with CD61,fibrinogen/fibro- nectin receptor CD42b Megakaryocytes gpIb, vWFreceptor CD43 T cells, myeloid cells, some B cell lymphomas CD45Panhematopoietic Signal transduction: tyrosine phosphatase CD45RA Bcells, naïve T cells Signal transduction: tyrosine phosphatase CD45ROMemory T cells Signal transduction: tyrosine phosphatase CD54Endothelium, activated cells Cell adhesion molecule, re- ceptor forCD11/CD18 integrin CD55 Most cells Inhibits complement activa- tion CD56NK cells Cell adhesion molecule CD57 NK cells CD59 Many hematopoieticcells, Blocks complement activity decreased or absent in PNH CD61Megakaryocytes gbIIIa, cell adhesion molecule with CD41, fibrinogenreceptor CD62P Activated platelets Cell adhesion molecule CD71Erythroid, lymphoid pre- Transferrin receptor cursors CD80 B cells,dendritic cells T cell costimulatory molecule CD86 B cells, dendriticcells T cell costimulatory molecule CD95 T cells Transmits apoptosissignal (similar to TNF receptor) CD103 Intestinal epithelial lympho-cytes CD122 T cells Interleukin 2 receptor beta chain

[0199] TABLE 2 Other exemplary antigens of hematologic interest AntigenCells Expressing Function HLA-DR B cells, monocytes, Class II MHC,antigen presentation myeloid progenitors, activated T cells IgM heavyNaïve B cells Antigen recognition, B cell activation chain (mu) IgGheavy Antigen-experienced Antigen recognition, B cell activation, chainB cells humoral immunity (gamma) IgD heavy Naïve B cells Antigenrecognition, B cell activation chain (delta) IgA heavyAntigen-experienced Antigen recognition, B cell activation, chain Bcells mucosal immunity (alpha) IgE heavy Antigen-experienced Antigenrecognition, B cell activation, chain B cells hypersensitivity reactions(epsilon) Kappa B cells Antigen recognition, B cell activation lightchain Lambda B cells Antigen recognition, B cell activation light chainTdT Immature lymphoid Ig and TCR rearrangement cells MiB-1/Ki-Proliferating cells 67

[0200] In a similar manner, the array format can be applied tocharacterizing other cell populations, such as the species and/orserotype of bacteria in a sample or the binding characteristics ofmolecules displayed by phage or yeast display methods. In the lattercase, antigens of interest may be immobilized at discrete sensorlocations on a sensor array, and the ability of cells displaying thecorresponding binding molecule bind to the antigen and result in achange in resonant frequency at a particular sensor location. A rankordering of affinities of display molecules can be obtained by titratingthe number of displaying cells applied to the sensor array.

[0201] Resonant sensors and sensor arrays also provide an attractivemeans to provide high throughput screening of molecules that bind tocell surface components such as receptors, or that compete with naturalligands for binding to these cell surface components. Conventionalscreening techniques typically yield hit rates in the 1-2% range onlibrary sizes of 1-3 million compounds. Thus, for a “good” primaryscreen, something like 15,000 compounds will be identified as “hits”.Secondary assays designed to eliminate compounds with adverse toxicity,adsorption, metabolic and elimination effects are applied together withelimination of compounds that bind irreversibly or non-specifically. Theend result is still hundreds of compounds with specific bindingproperties, very few or in many cases, none of which will prove to haveany clinical value. Such libraries of compounds can be used, togetherwith acoustic resonant sensors, and particularly resonant sensor arrays,with advantageous increases in screening efficiencies. The use ofvarious chemistries to protect, create hydrogels and/or attach compoundsor polymers to the surface of a microfabricated mechanical resonancesensor are described above.

[0202] Using such sensors, surface chemistries, and signal processing,one or more arrays (A₁ . . . A_(n)) may be created such that eacharrayed sensor in a particular array comprises one or more immobilizedmolecular species from a library of interest. A population of cells, orcellular material (e.g., proteins, lipids, enzymes, receptors, etc.) maybe contacted with this array, and binding of cells or cellular materialto the sensors in the array may be determined. One or more “standard”sensors, e.g., comprising a molecular species having a known interactionfor the cellular material of interest (e.g., a cell surface moleculepresent in the cell population), or comprising a molecular species knownnot to interact, may be included for normalization purposes.

[0203] Different arrays in an array “family” may comprise a defined setof properties, including, but not limited to: i) binding specificity,ii) binding affinity, iii) logP, iv) other properties indicative ofstructure/function relationships or v) combinations of these features.Subsequent arrays may be provided that exhibit a lesser, greater orsimply different property or properties than the previous and/orfollowing arrays. Using the data from screening each array in the“nested” family, and comparing that data to patterns from known targets,the researcher can quickly gain detailed insight into the identity andcharacteristics of the binding entity or entities. In addition,subsequent solution competition experiments with specific compounds onthe array surfaces can be easily performed to yield rapid informationabout binding specificity and affinity.

[0204] Exemplary applications for such high-throughput screening methodcan include kinase target discovery, e.g., using a kinase nested familyarray. In these embodiments, arrays comprising known kinase inhibitors,A(1) . . . A(n) immobilized at or near a sensor membrane, such thatmaterial binding to the kinase inhibitors (e.g., cells, proteins, etc.)will be within the sensed volume of the sensor are provided. Allinhibitors on A(1), for example, may be non-specific and have bindingaffinities to known kinases characterized by a dissociation constant(K_(d)) in the range of 10⁻⁷ to 10⁻⁹. A second array may then beprovided with inhibitors to known kinases having reduced bindingaffinities. Subsequent arrays may also be provided with differentbinding affinities or specificities to form the family of nested arrays.An expression product, cell population, tissue homogenate, or othertarget source may be contacted with A(1), and the binding pattern andaffinities recorded. This procedure may be repeated with subsequentarrays in the array family. A pattern (hit signatures) may be obtainedrepresenting known kinases. Subsequently, array A(1) may be contactedwith hits from A(2 . . . n) to determine competitive displacementkinetics. In a similar manner, Nested Family Arrays can be used toquickly distinguish and characterize unknown proteases, esterases,nucleases and transferases.

[0205] In another example, orphan G-protein receptors may be identifiedusing panels for comprising molecules known to interact with knownG-protein receptors families can be prepared and used to screen cellpreparations for possible orphan receptors. Such a panel would not onlyidentify the presence and quantity of receptor in the cell expressionmix, but also, by a resulting hit signature, assign a significant levelof information about the nature of the receptor binding site and it'sstructural similarity to other receptors in the G-protein receptorfamilies. In a like manner, other receptors, including nuclear hormonereceptors, may be patterned and characterized.

[0206] Another relevant property of cells relates to their subcellularstructure. Cells possess fibers which confer rigidity and the ability toexert forces on their environment. This includes but is not limited tointermediate filaments, microtubules, and contractile filaments such asactinomysin. In the case of contractile cells obtained from cardiacmuscle, individual cells can exert forces on the order of 1 micronewton.If attached to the surface of a sensor, these forces would be capable ofchanging the stress in the membrane and hence the frequency ofresonance. In addition to sensing the presence or absence of an attachedcell by density, resonant sensors and sensor arrays may be used to sensephysiologic differences in the strength of contraction (ionotropicchanges) and in the rate (chronotropic changes) of muscle contraction.

[0207] Measurement of these changes are very important in the functionof muscle, especially cardiac muscle, in which drugs are sought toaffect these two properties for the management of angina or congestiveheart failure. Thus, muscle cells cultured on a resonant sensor arraysubstrate may be queried with one or more molecules, and the resultingeffect on contraction determined. As stated above, the frequency ofresonance of the resonant membrane is highly sensitive to stresses inthe membrane. Changes in the force and rate of contraction of anadherent cell may be sensed by changes in the membrane resonance. In thespontaneously beating muscle cell, this will result in rhythmicmodulation of the fundamental frequency at a much lower frequency. Ahigh degree of resolution can be obtained in this spontaneously beatingsystem because this low frequency signal can be easily distinguishedfrom system noise. Inotropic changes will be seen as a greater degree ofmodulation where as chronotropic changes may be detected in thefrequency rate of the modulation. The unperturbed frequency of resonanceof the membrane is controlled by the method of deposition of themembrane, thickness and choice of materials. In the devices describedherein, a very thin layer (0.3 micrometer) of material with high Young'smodulus (silicon derivative) can be deposited under stress, to obtain amembrane with a membrane with a size and frequency range which minimizesdamping by water. A 1 micronewton stress provided by the cell would becalculated to cause an 80 Hz change in frequency. This is well withinthe limits of resolution of the devices described herein. In addition,the effect of the cell on the frequency of resonance can be increased bydecreasing the tension in the membrane. The high frequency of the devicecould be maintained by decreasing the diameter of the membrane, ifnecessary.

[0208] Once bound to a sensor array surface, either through specificinteraction with affinity agents or by simple use of a sensor arraysurface as a cell culture substrate, immobilized cells can provide anordered array for comparison of the biologic responses of various celltypes. Specific attachment may be mediated by cell surface molecule(s),which may be bound to a receptor that exhibits a sufficient bindingaffinity for the cell surface molecule(s). The term “cell surfacemolecule” refers to a molecule, or a portion of a molecule, present onan external surface of a cell. Preferred cell surface componentsinclude, but are not limited to, receptors such as pIgR, a scavengerreceptor, a gpi-linked protein, transferrin receptor, vitamin B12receptor, FcRn, integrins selectins, cadherins, N-CAM, I-CAM, lowdensity lipoprotein receptor; cargo carrier fragments such as pIgRstalk, members of the PGDF, FGF, and VEGF receptor families (e.g.,Flt-1, Flk-1, Flt-4, FGFR1, FGFR2, FGFR3, FGFR4), and surface antigens.The length of time for attachment of the cell to the array may belimited by capping and shedding or endocytosis of integral membraneproteins; however, such limitations can be overcome by a number ofprocedures. For example, assays could be completed quickly, e.g.,preferably within 2 hours of immobilization; more preferably within 1hour; and even more preferably within 30 minutes, thus limiting turnoverof membrane components. Alternatively, assays could be performed inunder conditions which slow or prevent membrane movement and/or turnover(e.g. at reduced temperatures or in the presence of endocytosisinhibiting agents such as EDTA). Another and preferred approach would beto provide additional sites for the cell to anchor to the sensor, suchas by mixing an integrin or other protein comprising an RGD containingpeptide with an antibody for a cell surface component. In this case,initial rapid immune mediated attachment would be followed by gradualspreading and attachment via integrins. The attached cells could then betreated with a variety of chemical or biologic agents, and theirresponse monitored.

[0209] A resonant sensor array comprising cells of interest may be used,for example, to determine the chemosensitivity and specificity ofchemotherapeutic agents In such an embodiment, an array of leukocytesobtained from a patient of interest may be immobilized by a panel of CDspecific antibodies. The panel would then be treated with increasingdoses of individual chemotherapeutic agents. The specific response ofthe leukemic cells could be measured by its selective detachment fromthe array, indicating selective injury or death to this cell type, andthus predicting toxicity (or lack thereof) in cells other than thetarget cells. In similar fashion, the addition of a biologic agent to aresonant sensor array comprising cells of interest may be used todetermine therapeutic specificity. Examples of this would be the testingof the specificity and toxicity of therapeutic monoclonal antibodies,aptamers, antisense, ribozymes or interfering RNAs.

[0210] Such a resonant sensor array comprising cells of interest mayalso be used to measure the susceptibility of related cells to viral,bacterial, or parasite infection. Such infection would result in changesin the physical properties of the cell (size, density, rigidity or celldeath) which would be detected by the sensor.

[0211] Another example for the use of resonant sensor arrays comprisingcells of interest is the addition of biologic agents to furthercharacterize a cell by the addition of specific immunologic reagents.This could be used for subset determination (e.g. through the use ofanother CD specific antibody to look at doubly positive cells), or tolook at activation state of the cell (e.g. to look at cytokine orchemokine receptor activation on the surface). Toxicity or apoptosis(e.g. using annexin V plus actinomycin-7AAD antibodies), endocytic orphagocytic activity (e.g., opson coated beads) could also be tested. Ineach of these cases, additional sensitivity could be obtained bycoupling a mass enhancement moiety to these secondary immunoreagents asdiscussed above.

[0212] In still another example, the ability of chemical or biologicagents to modify the number and or affinity of cell surface receptors onarrayed cells may be determined. For example, cells may be immobilizedto the array by binding to immobilized peptides with known affinitytoward the receptor. Addition of an agent which altered normal recyclingof the receptor, or affinity of the receptor (e.g., via phosphorylationand/or receptor dimerization) may result in displacement of the cellfrom the array surface. This is particularly useful, for example, in thecase of the opioid receptor, where some some opioid mimetics selectivelymodulate receptor turnover. Differences in these properties maycontribute to development of tolerance and addiction.

[0213] The skilled artisan will recognize that a commonly usedrefinement of the approaches discussed above is to create subregionswithin a particular resonant sensor array which allow small groups ofsensors to be exposed to different chemical or biochemical reagents. Asdiscussed in detail above, such a refinement facilitates more efficientand higher throughput screening of compounds for a variety of purposes.Molecules characterized according to their ability to bind at theresonant sensors may be further characterized by recovery and furtheranalysis, e.g., by mass spectroscopy, electrophoresis, microsequencing,NMR spectroscopy, etc. Additional advantages gained from the use ofresonant sensors and sensor arrays include the following:

[0214] Uses no molecular labels. Sensor surfaces in this invention areresponsive to mass binding. Immobilizing probe compounds, with knownbinding affinities to specific target families creates the ability topattern unknown targets, without labels that may affect the target'sbinding properties. As the sensor signal requires no fluorescent,isotopic or labels of any kind in preferred embodiments, the sample canbe presented to the sensor in native form.

[0215] Allows use of complex biological mixtures. Conventional assaysfor biological activity require that one or more binding partners bepurified and subsequently labeled with a molecular reporter capable ofbeing detected at relevant biological concentrations. As this sensor islabel-free, no purification may be required. The sensor surface detectsbinding only as determined by the biological specificity imparted by theprobe molecule. Thus, complex mixtures or even crude cell suspensionscan be presented to the surface without purification or samplepre-treatment of any kind. In addition to greatly simplifying andstreamlining the assay process, this capability allows fundamentally newinformation to be generated, as many protein targets cannot be purified.

[0216] Allows use of very small volumes. The sensor surface is verysmall (approximately 40 u) as is the array size (<1 cm2), requiring verylittle sample volume for a complete analysis. Our models indicate that acomplete sample analysis will require less than 20 μl of total samplevolume. This is a several thousand fold reduction over the best ofcurrently used techniques. Increased dynamic range by deep query and useof a hydrogel also leads to decreased crowding of ligands and hence lesssteric hinderance.

EXAMPLES

[0217] The following examples serve to illustrate the present invention.These examples are in no way intended to limit the scope of theinvention.

Example 1

[0218] Use of a Resonant Sensor as a Density Sensor

[0219] As described above, the device detects changes in densityconferred by concentration of biological molecules in the space abovethe sensor. Biological molecules such as protein (density 1.3) DNA(density 1.5) and RNA (density 1.7) are substantially higher than thatof water (density 1.0). and substantially higher than those of cells(density 1.05-1.09). However, when present in a monolayer, this altereddensity is barely seen because it only creates a very thin layer ofaltered density and the net density in the sphere is not substantiallychanged. The sensitivity of detection of the devices described hereinmay be amplified by creating a 3 dimensional region of binding (using ahydrogel ) which extends farther out into the hemisphere of waterinterrogated by the resonant membrane. The hydrogel-bonded resonantsensor is prepared as described in U.S. patent application Ser. No.10/306,506 (Atty Docket No. 035300-0502, filed Nov. 26, 2002).

[0220] An alternative approach to increase sensitivity is to couplesecondary labeling reagents agents to a mass enhancement moiety, such asgold beads, as was described above. As a first example, gold or magneticbeads are conjugated to one of 3 reagents: Anti-IgG, Anti-IgM orAnti-IgE. A small sample of a patient's serum is mixed separately witheach of these three, bead bound reagents. This Ig-class-specificimmunoaffinity reagent may be purified directly out of whole blood orserum merely by centrifugation or application of an external magnet.This can form the basis for a greatly simplified protocol for samplepreparation. These bead antibody complexes may then be applied directlyto the array. Sequential passage of IgE, IgM and IgG reagents across anarray of common allergic and infectious antigens could be used tocharacterize the allergic potential and immune status of a patient. Thisapproach could also be used to collect and quantify rare or secretedproducts.

Example 2

[0221] Use of a Resonant Sensor to Map Expression of Proteins

[0222] Antibodies immobilized to the resonant sensor may be used to mapthe expression of cellular proteins, much as nucleic acid “chips”currently are used to map the expression of cellular RNAs. In this case,an unlabeled protein extract is applied to a resonant sensor arraycontaining antibodies of interest embedded into a hydrogel. Thehydrogel-bonded resonant sensor is prepared as described in U.S. patentapplication Ser. No. 10/306,506 (Atty Docket No. 035300-0502, filed Nov.26, 2002). These antibodies may detect all forms of the protein, or maybe designed to detect specific conformers (e.g. phosphorylated forms).Binding of the protein confers a density shift, which may then beaugmented by addition of a mass enhancement reagent. This reagent couldbe used to distinguish conformational subtypes (e.g. a phosphorylatedform as above) or may be a natural dimerization partner. Thus allowingdeterminations of both structure (total amount of specific protein) aswell as function (ability to interact with other proteins). Changes inmass or density within the sensed volume above each sensor in the arrayare monitored by monitoring resonant frequency of the sensor membrane.

Example 3

[0223] Use of a Resonant Sensor to Assess Enzyme Activities

[0224] An array of enzyme substrates is produced by embedding thesubstrates in a hydrogel. The hydrogel-bonded resonant sensor isprepared as described in U.S. patent application Ser. No. 10/306,506(Atty Docket No. 035300-0502, filed Nov. 26, 2002). The resulting arrayis contacted with one or more enzymes of interest (e.g., kinases,hydrolases, esterases, etc.) and the resulting modification to eachsubstrate is detected by a mass conferring specificity agent (e.g. aphosphor-tyrosine specific antibody or a phosphor specific chelate.Changes in mass or density within the sensed volume above each sensor inthe array are monitored by monitoring resonant frequency of the sensormembrane.

[0225] An enzymatic activity sensor as described is used to detectchanges in mass conferred by enzymatic addition of mass to a substrate.In the case of DNA assays, the rolling circle, anchored SDA, anchoredPCR and anchored NASBA reactions may be monitored by such a sensor. Inthese cases, presence of a DNA species in solution is captured byspecific hybridization to a enzymatic activity sensor, where the enzymesubstrate immobilized at each sensor location is an oligonucleotidetarget. Amplification of nucleic acid sequences leads to the covalentaccumulation of considerable mass on the surface of the resonant sensor.

Example 4

[0226] Use of a Resonant Sensor to Assess Transcription FactorActivation

[0227] A resonant sensor array comprising mixed affinity reagents suchas nucleic acid transcription factor targets and antibodies to one ormore transcription factors is produced as described herein. Binding toantibodies would determine the total amount of transcription factorpresent and the amount and type of dimer partners available. Binding toDNA targets, would indicate the amount of functional protein present(e.g. phosphorylated transcription factor) as well as the dimer partnerchosen. Changes in mass or density within the sensed volume above eachsensor in the array are monitored by monitoring resonant frequency ofthe sensor membrane.

[0228] Thus, the present invention provides a sensor assembly andsensors with improved performance through a thermally insensitiveenvironment and short pathways for signals to travel to processingcomponents. Further, the modular construction for the sensors andhousing modules allows replacement of the sensors at a lower cost.Detection and analysis of the signals from the sensors is greatlyimproved through the use of the disclosed embodiments.

[0229] While preferred embodiments and methods have been shown anddescribed, it will be apparent to one of ordinary skill in the art thatnumerous alterations may be made without departing from the spirit orscope of the invention. Therefore, the invention is not limited exceptin accordance with the following claims.

We claim:
 1. A method of monitoring density in a fluid, comprising: placing said fluid into contact with a micromechanical sensor assembly comprising a membrane having an upper surface facing toward said fluid and a lower surface facing away from said fluid, wherein said membrane vibrates in response to electrical current; and monitoring a vibrational frequency of said membrane, wherein said vibrational frequency is related to density of a volume of said fluid at or near said membrane upper surface.
 2. The method of claim 1, wherein said fluid is an aqueous liquid environment comprising cells, and the density of said liquid environment changes due to a change in the number of cells in said liquid.
 3. The method of claim 2, wherein said cells increase in number due to division of said cells.
 4. The method of claim 2, wherein said cells are suspended in said liquid environment.
 5. The method of claim 2, wherein said cells are directly or indirectly affixed to said membrane upper surface.
 6. The method of claim 5, wherein said liquid environment comprises a hydrogel layer within the sensed volume of said micromechanical sensor assembly, and said cells are indirectly affixed to said membrane upper surface through said hydrogel.
 7. The method of claim 1, wherein said micromechanical sensor assembly is contained within a sensor array comprising a plurality of discretely addressable micromechanical sensor assembly sites.
 8. The method of claim 1, wherein said fluid is a gas.
 9. The method of claim 1 wherein the fluid contains dense organic or inorganic particles and the sensor is used to distinguish the mass or density of said particles.
 10. The method of claim 9, wherein said particles comprise heavy metals.
 11. The method in claim 7, wherein said density measurement is correlated to mobility of cells or cell processes across the surface of the sensor array
 12. The method of claim 2, wherein said cells are bacteria.
 13. The method of claim 2, wherein said cells are mammalian cells.
 14. The method of claim 13, wherein said mammalian cells are lymphocytes or hybridomas.
 15. The method of claim 2, wherein said cells are cells are yeast cells.
 16. The method of claim 15, wherein said yeast cells display antibody or antibody fragments, or protein libraries or protein fragment libraries.
 17. The method of claim 1, wherein said fluid is a liquid environment comprising virus or phage particles, and the density of said liquid changes due to a change in the number of virus or phage particles in said liquid.
 18. The method of claim 17 in which said virus or phage particles display antibodies or protein libraries on a particle surface.
 19. The method of claim 1, wherein said fluid is a liquid environment comprising molecules that bind to a receptor within the sensed volume of said membrane upper surface, and wherein the density of said liquid changes due to further addition of mass or density to the bound molecules.
 20. The method of claim 19, wherein addition of mass or density to the bound molecules comprises the use of an enzymatic reaction selected from the group consisting of strand displacement nucleic acid amplification, polymerase chain reaction nucleic acid amplification, isothermal nucleic acid amplification, or rolling circle nucleic acid amplification.
 21. The method of claim 19, wherein addition of mass or density to the specifically bound molecules comprises the use of one or more second binding partners for the bound molecules.
 22. The method of claim 21, wherein said one or more second binding partners comprise particles conjugated thereto.
 23. The method of claim 7, wherein said aqueous liquid environment is an electrophoretic gel within the sensed volume of an array of sensors, and said sensor array monitors flow of molecules through said electrophoretic gel.
 24. The method of claim 1, wherein said fluid is a liquid environment comprising molecules that bind to a second molecule within the sensed volume of said membrane upper surface, and wherein the method further comprises identification of molecules bound to one or more sensor sites by mass spectometry.
 25. The method of claim 1, wherein said fluid is a liquid environment comprising molecules that bind to one or more nucleic acid molecules within the sensed volume of said membrane upper surface, and wherein the density of said liquid changes due to binding of nucleic acid binding proteins to said one or more nucleic acid molecules.
 26. The method of claim 25, wherein said nucleic acid binding proteins are transcription factors or nucleic acid replication molecules.
 27. A method of screening a library of test molecules for the ability to bind to a population of cells, comprising: contacting an array comprising a plurality of discretely addressable micromechanical sensor assembly sites with said population of cells, wherein each site comprises a membrane having an upper surface facing toward an aqueous liquid environment comprising said population of cells and a lower surface facing away from said aqueous liquid environment, wherein said membrane vibrates in response to electrical current, and wherein each of said plurality of micromechanical sensor assembly sites comprise one or more test molecules from said library directly or indirectly affixed to said membrane upper surface; and determining whether a change in vibrational frequency of said membrane occurs at each of said plurality of micromechanical sensor assembly sites, wherein said vibrational frequency at each individual micromechanical sensor assembly site is related to binding of one or more cells to said one or more test molecules affixed at said individual micromechanical sensor assembly site.
 28. The method of claim 27, wherein said membrane upper surface comprises a hydrogel layer, and said one or more test molecules are indirectly affixed to said membrane upper surface through said hydrogel.
 29. The method of claim 27, wherein each member of said library of molecules is independently selected from the group consisting of small molecules, prodrugs, polypeptides, antibodies, antibody fragments, single-chain variable region fragments, polynucleotides, oligonucleotides, oligonucleotide analogs, oligosaccharides, polysaccharides, cyclic polypeptides, peptidomimetics, and aptamers.
 30. A method of screening a library of test molecules for the ability to inhibit binding of a ligand to a cell surface molecule, comprising: contacting an array comprising a plurality of discretely addressable micromechanical sensor assembly sites with a population of cells expressing said cell surface molecule and one or more test molecules, wherein each site comprises a membrane having an upper surface facing toward an aqueous liquid environment comprising said population of cells and a lower surface facing away from said aqueous liquid environment, wherein said membrane vibrates in response to electrical current, and wherein each of said plurality of micromechanical sensor assembly sites comprise said ligand directly or indirectly affixed to said membrane upper surface, and wherein each discretely addressable micromechanical sensor assembly site is contacted with different test molecules; and determining whether a change in vibrational frequency of said membrane occurs at each of said plurality of micromechanical sensor assembly sites, wherein said vibrational frequency at each individual micromechanical sensor assembly site is related to binding of one or more cells to said ligand affixed at said individual micromechanical sensor assembly site.
 30. A method of sorting cells in a cell population according to the expression of a plurality of cell surface molecules on said cells, comprising: contacting a sensor array with one or more test molecules that bind one or more cell surface molecules on one or more cells in the cell population, wherein said sensor array comprises (i) a plurality of discretely addressable micromechanical sensor assembly sites, each site comprising a micromechanical sensor assembly comprising a membrane having an upper surface facing toward an aqueous environment and a lower surface facing away from said fluid, wherein said membrane vibrates in response to electrical current, wherein a vibrational frequency of said membrane is related to density of a volume of said fluid at or near said membrane upper surface, and (ii) one or more cells from said cell population bound to an immobilized receptor for a cell surface molecule, thereby immobilizing said one or more cells within the sensed volume of each of said plurality of discretely addressable micromechanical sensor assembly sites; and monitoring a change in vibrational frequency of said membrane at each site resulting from one or more test molecules binding to said one or more cells within the sensed volume at each site.
 31. The method of claim 30, wherein a change in vibrational frequency at a site results from a change in a physiologic state of cell(s) bound at said site causing a change in size or density of said cell(s).
 32. The method of claim 30, wherein said method measures chemosensitivity of cells in said cell population to drugs, biological agents, pathogens, or environmental toxins.
 33. The method of claim 30, wherein said one or more cells are immobilized within the sensed volume of each of said plurality of discretely addressable micromechanical sensor assembly sites via binding to an integrin, selectin, cadherin, N-CAM, or I-CAM protein.
 34. The method of claim 30, wherein said one or more cells are immobilized within the sensed volume of each of said plurality of discretely addressable micromechanical sensor assembly sites via binding to an extracellular matrix protein.
 35. The method of claim 30, said method measures inotropic or chronotropic changes in cells in said cell population. 